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[Music]

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[Applause]

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I'm Tom dear from the University of

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Arkansas and I'd like to thank you all

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for coming and thank the organizers for

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the opportunity to present the work

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going on in our lab so sulfur specific

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maturation of nitrogenous in a

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methanogens I've got some to be pretty

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exciting results to share with you but

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first I want to go through a little bit

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of background so Earth's geochemistry

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has shifted pretty radically over

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geologic time early on we think there

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were locally abundant sources of reduced

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iron and sulfide and that ancient cells

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broadly made use of these and

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incorporating clusters into iron sulfur

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proteins to carry out important

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reactions for life and with the rise of

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atmospheric oxygen these became much

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less attractive cofactors so where

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possible Arabs have minimised their use

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but modern anaerobes particularly strict

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Anna ropes like methanogens still make

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use of loads of higher and sulfur

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proteins and so they sort of represent a

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window into the past you know get some

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methanogens are also extremely important

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in studying the nitrogen cycle thanks to

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some really great work by eric boyd and

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john peters and many others there's good

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evidence that the nitrogenase enzyme

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that's responsible for the biological

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fixation of dinitrogen first arose in

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methanogens so a broad outline of the

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way that the the reaction progresses

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dinitrogen is reduced to ammonia at the

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expense of reducing equivalents and

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quite a bit of energy and there are iron

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sulfur clusters some somewhat bizarre

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ones compared to the broad range of iron

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sulfur proteins that are required

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to carry this out and then methanogens

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notably code for a lot of other iron

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sulfur proteins that are of interest to

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astrobiologists and people studying

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early Earth including Farah dachshunds

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hydrogenase and carbon monoxide

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dehydrogenase but given that all of

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these iron sulfur clusters are needed in

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methanogens we know little to nothing

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about how they're actually being

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assembled so what we do know about iron

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sulfur cluster biogenesis we mostly know

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from bacteria and eukaryotes so there

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are three described systems with some

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commonalities running through them

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the first system described was actually

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the NIF system this is specific for

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nitrogenase described in his Oda dr. van

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LAN di next described was the is C or

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ystem and so this is quite similar to

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NIF but more widely distributed among

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bacteria and the mitochondria of

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eukaryotes this is more of a

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housekeeping system for general iron

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sulfur cluster biogenesis and then last

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described was the sough system and this

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is especially important today in strict

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anaerobes strict anaerobic bacteria and

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in the chloroplasts of plants common to

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all three systems is the overall

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reaction scheme which is a cysteine d

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sulfurous to take sulfur from cysteine

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and then to help assemble with some iron

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coming from somewhere it varies

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depending on the organism in the system

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to assemble a cluster on a scaffold

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protein so for NIF and ISC the cysteine

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t sulfur aces are the S proteins the u

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proteins of the scaffolds and then from

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the scaffold the cluster can be handed

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off to a target able protein the

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subsystem works the same way in it's

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been described in bacteria but the

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scaffold is a multi mer of B C or B C

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and D soft proteins but all of

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have in common the facts that free iron

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and sulfide are broadly toxic to most

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cells so controlled biogenesis is

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required and for all three of these

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systems as described everything kind of

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runs through cysteine cysteine is the

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sulfur donor now methanogens

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categorically seem to lack an if type

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dedicated biogenesis system for

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nitrogenase which is sort of surprising

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because you can find the the core

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nitrogenous genes in members of all

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seven orders of extant methanogens the

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two deeply rooted orders are somewhat

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metabolically restricted to co2

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reduction these do not use cysteine as a

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soul sulfur source when they're growing

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and there actually is some evidence

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showing that inorganic sulfide is the

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direct donor for iron sulfur clusters so

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that flies in the face of what I just

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showed you for everything we know from

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bacteria and eukaryotes so they are not

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predicted to code for a cysteine sulfur

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race just the course of proteins and

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I'll go ahead and tell you now those

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soft proteins seem like they're

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universally conserved in methanogens and

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even more broadly in archaea so the five

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later evolving orders of methanogens so

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also have those soft core proteins many

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of them also appear to have the core

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misc system as well these are more

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flexible they some of them are capable

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of multiple methanogenesis pathways and

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have briefly been reported several of

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them to be able to grow on cysteine or

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sulfide as a sole sulfur source and for

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some of them even more compounds than

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that in our lab we mainly work with

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members of the genus Mathias Arsena

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order matheno sarsen Allie's and I'm

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gonna tell you a little bit more about

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the one that we love the most next so

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matheno SAR saina heceta for ends or

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asset difference depending on who you're

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talking to is a metal metabolically

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flexible model meth antigen it's a great

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model system

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for one thing there's a strong genetic

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system that's been developed so that we

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can manipulate the genome and also it

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just has lots of copies of everything so

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there are as many as maybe three copies

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of the ISC system two copies of the

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subsystem and this is sort of unusual if

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you look in bacteria to have many copies

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of things but it looks like it's not the

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only method of SARS on a species that

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has this and we can confirm that it can

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grow on cysteine or sulfide or a

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combination of the two sulfur sources

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and does just fine

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it also appears to have all three known

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versions of nitrogenous molybdenum

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vanadium and the iron only nitrogenase

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so the molybdenum nitrogenase is the one

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you find in all desert ropes the

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vanadium and iron only nitrogenases are

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the alternative nitrogenases and i'm not

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going to talk any more about them today

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so from here forward I'll be talking

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about the molybdenum nitrogenase and I

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started first looking at the is c2 gene

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cluster these are the proteins that have

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been detected at the highest levels most

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consistently in proteomic studies of a

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messy divorce

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so I briefly I cloned the two core

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components the cysteine d sulphurous and

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the scaffold proteins s2 and u 2 into e

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coli overexpressed purified to

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homogeneity and i did the same for a

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massive runs as a Cana tastes just to

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have a physiologically relevant target

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protein to load cluster into so we can

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build cluster on the scaffold you

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protein using the S protein and then

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incubated with April a Cana tastes and

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monitor for activity and what we see is

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we only get robust activation of April

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that kind of taste when it's being

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incubated with cluster loaded scaffold

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so on its own there's basically no act

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and if we incubate it with equivalent

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amounts of iron and sulfide in a

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reducing environment to what would be

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found on the scaffold we basically have

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no activity again so in vitro this looks

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like a bonafide iron sulfur cluster

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biogenesis system so we can knock out

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these genes and we did that using

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essentially standard techniques and what

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we see is moderately impaired growth

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when this mutant has grown with cysteine

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as the sole sulfur source there's a

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little bit of variability from

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experiment to experiment but

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consistently the mutant prefers sulfide

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we can actually a say for cysteine T

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sulfurous activity in sulfur eli sites

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and when we do that we see significantly

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reduced cysteine d sulphureus activity

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and the mutant compared to the parent

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regardless of what Sulphurs horse the

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cells groove on so we see what we think

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is some altered sulfur metabolism

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although there's no gross cluster

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assembly defect so if we just look at

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acid labile sulfide to see how much

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cluster content there is and the mutant

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versus the parent there's no significant

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difference regardless of sulfur source

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when we look at sulfate sulfur so this

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is mechanistically important for the

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cysteine ki sulfurous it's sort of how

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it works and this is also has

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implications for the sulfur relay system

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in sulfur metabolism when we look at

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sulfate and sulfur we see a significant

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reduction

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anytime cysteine is present as a sulfur

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source so that's sort of an interesting

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thing to see in combination with

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basically no difference in cluster

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content so switching back to the

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wild-type real fast a massive difference

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can fix nitrogen regardless of what it's

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using as a sulfur source cysteine versus

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sulfide maybe somewhat surprisingly

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neither cysteine nor alanine which is

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the sort of side product of 16t sulfur

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ace neither one of these amino acids

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by having an amino group can serve as a

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nitrogen source for in assitive wrens so

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when we grow under nitrogen replete

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conditions or under nitrogen fixing

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conditions where there's no ammonium

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present there doesn't seem to be a huge

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difference our preference for sulfur

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source and when we do a Western blot and

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go looking for the catalytic subunit one

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of catalytic subunits of the molybdenum

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nitrogenase we see it show up only when

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ammonium is absent and it's being

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produced abundantly whether cysteine or

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sulfide is the sulfur source so the

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mutant strain deleted of ISC 2 when we

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grow it under nitrogen fixing conditions

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with sulfide as a sulfur source there is

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no phenotype whatsoever however if we

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try growing it with cysteine there is a

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pronounced growth defect again that

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varies somewhat from experiment to

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experiment this is probably the most

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severe iteration of it that we've seen

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but there is a strong strong preference

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for growth with sulfide as the sulfur

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source under nitrogen fixing conditions

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especially with this mutant so that has

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led us to a proposed model of cluster

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biogenesis in the methanogens M assitive

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runs in which cysteine is routed through

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the ISC system and then can be used to

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load cluster into proteins as needed and

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that inorganic sulfide may be going

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through perhaps a sort of primordial

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subsystem because these subsystems I

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told you they're pretty universal in

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archaea but they're never really paired

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with a suffice cysteine to sulphurous so

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it seems like the subsystem is maybe

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primordial in archaea and and not really

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geared towards using cysteine so the

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results that i've showed are consistent

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we are still being able to load cluster

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in

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but there is a serious problem once we

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cut off the ISE system and that specific

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is c2 is C 1 and is C 3 are still

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present but apparently they are not able

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to provide for normal maturation of the

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molybdenum molybdenum nitrogenase when

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cysteine is the sulfur source and so I'd

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like to thank my advisor P I dam Lesnar

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other members of the lesner lab our

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collaborator ever doin who's done some

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great biophysical characterization of

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proteins for us that I didn't really

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have time to share with you today and

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then also thank the funding agencies for

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their generosity and with that I'll take

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any questions thanks Thomas you have

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time for a few questions yeah that was

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really nice he mentioned that your

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bacterium has all three different types

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of nitrogenases i didn't can't perhaps

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already said this but are you looking at

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a deleted version that only has one at a

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time or have you tried building such a

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thing - I see whether some of them might

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be more affected by the by the various

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conditions that you've been describing

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we are looking into that we don't have

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anything where we have actually deleted

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the nitrogenase but it's possible to

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grow under conditions where you don't

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give them any molybdenum and then they

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have to use alternative nitrogenases so

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I think we have time for other questions

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if there are any thanks for the great

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talk and the data were coming pretty

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quickly so I'm sorry if I oh sure miss

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something but it seems like in the is

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situ mutant eat undergrowth with

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sulphide you had and maybe even an

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increased amount of sulphate sulphur and

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you also saw some sort of ISC to like

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activity does that suggest there's

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another group of proteins that are

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accomplishing this function yes

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certainly for the second part of your

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question the most parsimonious

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explanation would be those other is see

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clusters have bonafide cysteine t sulfur

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races a more complicated answer would be

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in the methanogens that we don't really

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work with and that are predicted not to

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have cysteine to sulfur ace if you crack

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them open and look for assisting to self

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race activity you'll find it so we

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probably don't know everything that's

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going on I think it would be really

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great to look at that latter at class of

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methanogens with these assays mm-hmm

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it's really exciting Thank You Thomas

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all right thanks so our next speaker

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Miriam accessibility is she is around so

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it doesn't look like she might have made

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it to the conference so I apologize

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after that talk isn't gonna be coming up

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so we have a few minutes if there's

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other questions or waiting for the next

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talk for the speakers that have already

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presented happy to field a few questions

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Chirk Shawn I have a question for you

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yeah go home before the oxygenation of

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the atmosphere what would be the source

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of sulfite I I kind of thought that it

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went to either sulfate or sulfide but is

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there a sulfite component and what we

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expect to be coming out from volcanoes

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and then going into the ocean yeah so I

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won't bring it up but you know sulfur

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dioxide so I mentioned the disproportion

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ation reaction where it goes to sulfate

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as well as hydrogen sulfide but that's

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at higher temperatures at lower

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temperatures that can just hydrate

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it's a sulfuric acid as well with water

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and so that can specie 8 depending on

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you know the pH it could be at lower pH

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sulfur dioxide or sulfuric acid at high

362
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rpm I so fight already

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still higher pH sulfite so there's a

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potential that you could have sulfite

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there and without oxygen or ferric iron

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present it could potentially be stable

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and I had a diagram on an extra slide

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sure I think there's a question in the

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I was so we know that for example in eco

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light the soft system is assigned for

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the de novo cluster synthesis compared

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to IOC which is basically for repair

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when one iron falls off you just use ISC

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to put that one on and back and like in

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the lab for example we we put our enzyme

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is C D G G for reduction and iron and it

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basically repairs the cluster without

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the need for anything else so is there

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anything similar like these two jobs for

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like are the two in your box doing the

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same thing or they are all like doing

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just then although synthesis not

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entirely short so the the very first

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part of what you were saying that the

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different jobs different roles for ISC

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and stuff and E coli yep

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so my understanding is that is C is that

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the general system and stuff is more

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expressed if there is oxidative stress

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or a lack of iron is that the other way

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round but yeah okay yeah I was just

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wondering if that's the case here or

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they are just like two systems doing

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exactly the same job probably not doing

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exactly the same job so if you were

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talking about a little bit of oxidative

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stress

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we're like one iron has fallen out of

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the cluster then yeah I would guess that

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probably either one could could handle

401
00:20:09,780 --> 00:20:08,530
that kind of repair but well and for

402
00:20:12,660 --> 00:20:09,790
instance with the connotates if you

403
00:20:14,400 --> 00:20:12,670
don't really make it a Poe and fully

404
00:20:16,890 --> 00:20:14,410
strip it out usually what's happening is

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one irons being lost and you can

406
00:20:22,399 --> 00:20:19,020
reactivate it like pretty easily just by

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incubation with DDT

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so yeah that probably doesn't answer