Bacterial DRAMA: Dispute Results in an Anti-Microbial Agent

Part 1: Dylan McClung, PhD Interview

Duration: 00:10:17
Transcript

00:00:06:01 - Hi I'm Thi Nguyen with the|Science Communication Lab.
00:00:08:20 This year we got to talk|to all the presenters
00:00:10:17 " for ""Share Your Research."""
00:00:11:18 I'm so glad we get to hear their voices
00:00:13:10 and learn a little bit more about them.
00:00:14:18 Today we're talking to Dr. Dyna.
00:00:16:14 Dr. Dyna could you introduce yourself?
00:00:19:03 - Of course.
00:00:20:03 Hi my name is Dylan McClung|AKA Dyna Cockus Rose.
00:00:24:15 I'm a microbiologist and a drag queen
00:00:27:09 and I study bacteria that live in the soil
00:00:30:04 and I used the interactions|that bacteria have
00:00:36:04 to discover a brand new antibiotic|that I named Dynaplanin.
00:00:40:06 And all of this was part of my PhD work
00:00:42:19 at the University of California Berkeley
00:00:44:16 where I recently graduated.
00:00:46:15 - And Dyna what motivated|you to get into science?
00:00:50:17 - Great question.
00:00:52:23 I feel like I've always wanted|to help people in some way.
00:00:58:11 I originally wanted to be a medical doctor
00:01:01:00 but I realized pretty quickly
00:01:03:14 that I cannot handle blood of any sort.
00:01:06:15 So when I was doing my|biology major in college
00:01:12:02 at The College of New Jersey
00:01:13:18 I started pivoting more towards research
00:01:16:17 and understanding what|a research lab was like.
00:01:20:13 And one of the new faculty|her name is Dr. Katie Elliot
00:01:23:10 she worked on bacteria and did genetics
00:01:26:05 " and I thought ""Ooh bacteria cool."
00:01:28:02 " Like that's medical."""
00:01:30:17 Turns out we did nothing|at all medical in the lab
00:01:33:23 but it really developed|I developed this passion
00:01:40:03 for microbiology inside this|lab and realized that there's
00:01:45:03 so much that microbes do that|we don't really appreciate.
00:01:49:19 - As you started getting into research
00:01:51:12 and looking at some of these questions
00:01:53:07 what is an early discovery that you made?
00:01:56:03 (Dr. Dyna laughing)
00:01:57:13 - Well I mentioned my|college research lab
00:02:01:05 and Dr. Katie Elliott's lab
00:02:03:09 and we were studying some|genetic components particularly
00:02:07:11 I was interested in how|this one gene called yqgF
00:02:12:00 how it might function within cells.
00:02:14:04 We didn't really know it was|called a hypothetical protein.
00:02:17:21 And so I was studying how it|might relate to DNA repair
00:02:25:06 and fixing some breaks within the DNA.
00:02:30:07 And part of that process is doing
00:02:33:21 an experiment called a|transformation frequency experiment
00:02:37:02 where we can add some|DNA and give it to cells
00:02:40:08 and measure how many|cells actually take up
00:02:42:09 that DNA and put it onto their chromosome.
00:02:45:23 And I was running this|experiment for months
00:02:49:12 and my results were|not coming back the way
00:02:52:12 that my advisor expected them to
00:02:55:03 and she was very confused|and she kept just asking me
00:02:57:17 to repeat these experiments.
00:02:59:19 And I realized...
00:03:02:07 It ended up coming down to her|watching me from the start
00:03:06:11 do the entire experiment
00:03:07:17 from streaking out...
00:03:09:09 Excuse me streaking out|strains from a freezer stock
00:03:13:00 all the way to like performing it.
00:03:15:03 And as I was streaking out|these stocks from the freezer
00:03:20:22 she realized I was using the wrong tube
00:03:24:02 as my reference the entire time.
00:03:27:07 And I didn't realize because
00:03:30:01 there were actually two different strains
00:03:31:23 that we call wild-type meaning unmutated
00:03:35:17 kind of a reference standard
00:03:37:23 and one of them was for|a particular collection
00:03:41:06 and another was for a|different collection.
00:03:43:13 And so what I learned is
00:03:44:14 there's something called isogenic strains
00:03:47:01 meaning that you need to have|exactly one mutation different
00:03:50:19 between your strains to compare them.
00:03:52:21 And these wild-types were not isogenic
00:03:55:21 they had lots of different mutations
00:03:57:15 so I needed to focus on just|the one group at a time
00:04:00:17 and this other group at a time|and had to repeat basically
00:04:04:23 a year's worth of experiments|in like one month.
00:04:07:20 (Dr. Dyna laughing)
00:04:09:16 But it taught me to be very...
00:04:13:13 Pay attention to what's|going on and figure out
00:04:16:09 what exactly I'm working|with at all times
00:04:19:03 so that I'm not making crazy|mistakes like that again.
00:04:22:08 - Yeah research is all|about learning from mistakes
00:04:25:12 and from experiments|that didn't work right?
00:04:28:21 So.
00:04:30:02 - Yes yes.|- Speaking of adding DNA
00:04:32:20 it looks like you've added|DNA to your outfit today.
00:04:36:00 Could you tell us a little bit about this?
00:04:38:09 - Of course. Yeah.
00:04:39:08 So my drag persona Dyna Cockus|Rose is all about science.
00:04:44:01 I'm super inspired by the work|that I do as a researcher
00:04:46:14 and I wanted to incorporate|that in a more creative way
00:04:49:12 and communicate science|through the art of drag.
00:04:52:16 So Deinococcus is actually the|group a genus of organisms.
00:05:00:05 The most famous is called|Deinococcus radiodurans
00:05:03:06 " which is actually in the|""Guinness Book of World Records"""
00:05:05:14 for most radiation resistant lifeform.
00:05:08:12 And I thought Dyna and Dylan|sounded kind of similar
00:05:12:05 I kinda connected to that
00:05:13:10 so I chose Dyna Cockus as|my first and middle name.
00:05:17:08 Rose actually comes from|my first drag mother
00:05:19:16 who was the first person|who put me into drag
00:05:21:23 kind of a mentor in the drag scene.
00:05:24:23 And so her last name is Rose|her full name's Sandra Rose.
00:05:29:10 So I try to incorporate|science into my drag
00:05:32:11 as much as possible.
00:05:33:16 So this is a DNA bodysuit
00:05:35:14 I'm kind of like a|chromosome got my two arms
00:05:38:19 my two legs like a human chromosome.
00:05:43:05 Yeah. Yeah.
00:05:44:04 I wanted to represent DNA in|a fashionable and fun way.
00:05:48:10 - You've done it really well.
00:05:50:11 As...|- Thank you.
00:05:52:03 - A person in science|who expresses yourself
00:05:55:19 in a way that incorporates science
00:05:58:01 what is one thing that|you want people to know
00:06:00:09 about being a scientist|that they may not know?
00:06:02:22 - I think something that I|always get as a drag performer
00:06:08:05 when I tell people that|I'm a scientist is
00:06:11:02 " ""Ah well that's boring."""
00:06:12:23 " ""Ah that's so dry."""
00:06:14:23 And I don't see that at all right?
00:06:17:08 I find science fascinating.
00:06:19:04 I find learning about|the world so intriguing.
00:06:22:17 And so I wanna try to present that as much
00:06:25:03 as possible with my drag character
00:06:27:22 and what I want other|people to understand is
00:06:30:09 that science and scientists|in particular are so diverse.
00:06:34:20 There are so many|different kinds of people
00:06:36:09 there are people like me who do drag
00:06:39:01 I'm not the only drag|artist who's a scientist.
00:06:42:22 There are some people|who are really athletic
00:06:45:04 some people who are really personable
00:06:46:22 some people who are really shy
00:06:48:22 and like the video games|or board games and things
00:06:51:23 but there are so many|different kinds of people
00:06:54:13 who do science for a variety|of different reasons.
00:06:57:22 And I think all of these people|are great and really cool.
00:07:04:01 (Dr. Dyna laughing)
00:07:04:21 So I don't see us as boring
00:07:07:16 and so I want people to realize
00:07:09:20 that we are complex people|who have our own personalities
00:07:13:12 and we're not all cookie cutter.
00:07:17:12 - Yeah and it all|contributes to the science
00:07:19:10 that we do too.
00:07:22:00 If you...|- Absolutely.
00:07:23:03 - If you encountered a young|person who wanted to learn
00:07:26:23 about getting into science|and being a scientist
00:07:29:11 in your field what are|some of the greatest things
00:07:32:08 that you would tell them?
00:07:33:06 Or what would you tell them about some
00:07:34:16 of the greatest things in your field
00:07:36:03 and some of the challenging|things that they can expect?
00:07:39:08 - So I work in particular on a group
00:07:42:05 of bacteria called the rare actinomycetes
00:07:45:06 which are under studied bacteria.
00:07:46:20 They mostly live in the soil.
00:07:48:10 And the one I study in particular
00:07:49:22 is called Couchioplanes caeruleus.
00:07:52:15 And what is great about these microbes
00:07:56:02 like I talked about in my presentation
00:07:58:18 is that there's a lot of|potential for them to produce
00:08:01:16 these natural product molecules|that could be antibiotics
00:08:05:02 or antifungals.
00:08:06:02 There's tons of opportunity|to find new drugs
00:08:10:06 and medicines for us which|is the very exciting part
00:08:13:04 about the field in addition|to all the complex interactions
00:08:17:08 that they might have in nature.
00:08:19:19 But what is particularly challenging is
00:08:22:11 that they are so understudied.
00:08:24:17 So while there's a lot of opportunity
00:08:26:23 there's also a lot of challenges.
00:08:29:02 It's kind of a double edged sword.
00:08:30:14 So for reference Couchioplanes caeruleus
00:08:33:19 had exactly nine papers|published about it
00:08:37:05 I will have published the 10th|paper once mine goes live
00:08:43:01 and E. coli for example has|400000 papers published on it.
00:08:48:11 So there's a lot of|information that we don't know
00:08:51:05 about Couchioplanes which|really limited what I was able
00:08:54:07 to accomplish with this bacterium.
00:08:57:01 But again super exciting|because there are a lot
00:08:59:20 of unanswered questions
00:09:01:03 and we might find something really cool
00:09:02:20 that nobody has ever discovered before.
00:09:05:00 - That's really excitement
00:09:05:22 this idea of publishing|the 10th paper in a field.
00:09:11:04 - Yeah.|- You're really contributing
00:09:12:07 new knowledge.
00:09:13:11 - I think that's one of the coolest parts
00:09:16:12 about doing a PhD honestly
00:09:18:07 is I realized at some point|during maybe my fourth
00:09:22:10 or fifth year that like I always knew
00:09:25:20 that research was about discovery
00:09:27:19 but I never thought|about it in the terms of
00:09:30:07 I am creating new|knowledge for somebody else
00:09:33:20 to then learn from and build off of
00:09:36:01 which I think was super empowering
00:09:38:01 and really exciting and|something that I needed during
00:09:40:14 the hard research times|I was going through.
00:09:44:06 - So if you're tuning in
00:09:45:13 please check out Dyna's presentation
00:09:48:07 " of ""Share Your Research."""
00:09:49:14 Thank you so much for joining|us and talking with us today.

Part 2: A newly discovered antibiotic

Duration: 00:19:46
Transcript

00:00:12:15 - Hi my name is Dyna Cockus Rose.
00:00:14:21 I'm a drag queen and a microbiologist and I love drama.
00:00:19:05 And it turns out there's drama
00:00:20:16 all around us on the microbial scale.
00:00:23:18 Anything from bacteria arguing over resources
00:00:27:05 like tip money or nutrients to very serious threats
00:00:31:05 like the rise in antimicrobial resistance
00:00:33:20 which the World Health Organization predicts
00:00:35:21 will be the leading cause of death by 2050.
00:00:39:00 So that means the antibiotics that we have now
00:00:41:01 are not gonna be as effective in 30 years.
00:00:43:16 And we already know that they're not as effective
00:00:45:11 as they were 30 years ago.
00:00:47:16 So we need to find new medicines
00:00:50:13 new therapeutics now to start combating that issue.
00:00:54:15 And that's where I come in.
00:00:56:20 So I went to a very glamorous place
00:00:59:18 to start my search the soil.
00:01:03:18 I know you might be thinking
00:01:04:19 " ""How did she find an antibiotic in soil?"""
00:01:08:11 Well let's dive in.
00:01:10:19 Turns out that soils have tons of microbial diversity
00:01:15:18 anything from bacteria to fungi
00:01:18:07 and even microscopic animals but one characteristic
00:01:21:17 about soil is that it's really nutrient limited.
00:01:24:20 So that means there's a lot
00:01:25:18 of competition for those nutrients and space.
00:01:28:21 And of course I love a good competition.
00:01:31:21 So I wanted to study this in the lab.
00:01:35:01 But of course bacteria
00:01:37:01 they have to defend themselves using chemicals
00:01:40:15 because they don't have things
00:01:41:17 like nails or claws or anything like that.
00:01:45:15 So I'm studying these chemical defense molecules
00:01:48:11 for my PhD work but we wanna simplify
00:01:50:23 this a little bit in the lab.
00:01:52:16 So I'm only gonna focus on three players.
00:01:54:16 A red and a blue bacterium that are competing for resources
00:01:58:02 and then the yellow bacterium which is a bystander.
00:02:01:00 Now this is in the lab used as an indicator strain
00:02:04:21 meaning that it indicates whether or not
00:02:06:20 an antibiotic is produced.
00:02:08:18 So if the blue and red bacteria are competing for resources
00:02:12:23 the blue one might respond by producing an antibiotic.
00:02:15:18 It's gonna make this molecule in its cells
00:02:17:17 and secrete it outside
00:02:19:12 and then that molecule will diffuse away.
00:02:21:18 So that means there's going to be a high concentration
00:02:24:05 of antibiotic close to the cell
00:02:26:05 and a lower concentration away.
00:02:28:13 And any bacteria that are sensitive are gonna end up dying.
00:02:32:05 And in a lab we call that a zone of clearance
00:02:34:07 because we see a transparent clearing zone
00:02:36:21 where no bacteria are growing.
00:02:39:04 So my question for my PhD work
00:02:41:08 was can I use this process that's happening
00:02:44:09 in nature to find a new antibiotic in the lab?
00:02:48:08 So the bacteria that I study are called actinomycetes.
00:02:51:10 They love to grow in the soil
00:02:53:06 and they come in a variety of shapes sizes and colors
00:02:56:18 but they are experts at chemical defense
00:02:59:04 and that's why I'm particularly interested in them.
00:03:02:03 They make a general class of molecules
00:03:04:15 called natural products that have a variety
00:03:07:00 of biological activities from antibiotics
00:03:09:23 to antifungals and even anti-tumor agents.
00:03:12:23 And it turns out that the actinomycetes
00:03:14:18 are responsible for about 70% of the natural products
00:03:18:04 that we've currently discovered.
00:03:20:03 Can you believe that?
00:03:21:04 Bacteria were the first ones to make these antibiotics.
00:03:26:00 So most of them were produced by streptomyces
00:03:29:00 and that's the most well studied group
00:03:31:10 or genera in the actinomycetes.
00:03:33:16 But today I'm gonna focus on the rare actinomycetes
00:03:36:14 because they're under studied a little bit more unique
00:03:39:08 and there's a lot of potential for new discovery.
00:03:42:13 Based on DNA sequencing we know that there are greater
00:03:46:02 than 99% of the natural products
00:03:49:05 in the rare actinomycetes that are still undiscovered.
00:03:51:20 So that's a lot of molecules that we can use
00:03:54:12 for healthcare and a variety of other properties.
00:03:57:21 So I'm gonna split my talk today
00:03:59:08 and answer four major questions.
00:04:01:05 One what method did I use?
00:04:03:01 How did I discover this antibacterial?
00:04:05:17 Two what is it?
00:04:06:14 So what's its chemical structure?
00:04:08:17 Three what genes are necessary?
00:04:11:03 How is it made?
00:04:12:12 And four how does it work?
00:04:14:14 What are the ways that it kills a bacterial cell?
00:04:18:18 So let's start with how I found them.
00:04:21:07 I mentioned that I study these rare actinomycetes.
00:04:23:19 These are bacteria that are under studied in the lab
00:04:27:14 basically any kind of soil dwelling microbe
00:04:29:23 that's not streptomyces.
00:04:32:16 And I chose eight of them that are from different subgroups
00:04:38:15 called genera so that I could increase the diversity
00:04:41:20 of species that I was working with.
00:04:44:02 And this entire point of this workflow
00:04:46:12 is to increase the chances
00:04:47:19 of finding a brand new antibiotic.
00:04:51:06 So they may have these antibiotics
00:04:53:05 but one question in the field is how do we turn
00:04:55:16 on the genes that are responsible
00:04:57:07 for making those antibiotics?
00:04:59:01 The way our lab does it is using ecology.
00:05:01:13 So we make bacteria interact
00:05:03:14 or potentially compete with each other.
00:05:06:05 So in this case I might have the red
00:05:07:21 and blue bacterium growing on their own Petri dishes.
00:05:10:15 And I would not expect them to produce antibiotics
00:05:13:02 because there's no competition happening.
00:05:15:05 But if they're both grown on the same Petri dish
00:05:18:00 there might be competition for resources
00:05:20:00 and one bacterium might produce an antibiotic
00:05:23:13 in an attempt to kill the second one.
00:05:26:14 So if I put all eight of these bacteria
00:05:29:09 into every pair wise combination
00:05:31:00 that's a total of 28 unique interactions
00:05:33:16 that I had to screen through
00:05:35:03 which is actually a pretty small pilot screen.
00:05:38:07 And at this point we don't know which bacteria
00:05:40:18 combinations are actually gonna result in an antibiotic.
00:05:43:16 So that's why we wanna do all of them possible.
00:05:47:08 And then the third piece is we overlay
00:05:49:14 with that indicator strain.
00:05:51:02 In this case I'm using a multi-drug resistant bacterium.
00:05:54:13 So the way this works is I take this indicator strain mixed
00:05:58:03 in a liquid and I spread it over top
00:05:59:22 of that Petri dish completely submerging the colonies.
00:06:03:01 And if that bacterium grows we'll see kind of a hazy
00:06:05:22 beige-ish color over top of everything.
00:06:08:17 If there's an antibiotic produced
00:06:10:07 we'll see a zone of clearance
00:06:11:13 like the one around the red bacterium in this image.
00:06:14:22 The bacterium that I chose as an indicator strain
00:06:17:04 is called Amycolatopsis species AA4.
00:06:19:17 So it is resistant to at least 15 known antibiotics
00:06:24:02 and that's why we chose it.
00:06:25:11 So if we can find an antibiotic that kills AA4
00:06:28:10 we know it's not one of those 15
00:06:30:01 and probably not even related to one of those 15.
00:06:32:12 Again increasing our chances of finding something new.
00:06:35:15 And then we can scale up this process
00:06:38:03 purify the antibiotic and figure out all of its properties.
00:06:41:19 So I told you I screened 28 of those interactions
00:06:46:01 and there was exactly one candidate that fit the bill.
00:06:49:22 And that was shown here between these two bacteria
00:06:53:16 that are numbered T137 and T141.
00:06:57:00 So there are two colonies
00:06:58:17 the orange and yellow spots in the middle.
00:07:01:09 Each colony is about a million
00:07:02:17 to a billion bacterial cells all clumped together.
00:07:05:07 And this was the only one it's only combination
00:07:07:22 that produced a zone of clearance.
00:07:09:21 And these bacteria are called Actinoplanes durhamensis
00:07:13:03 and Couchioplanes caeruleus.
00:07:14:20 And we found that the colony on the right Couchioplanes
00:07:17:11 is the antibiotic producer.
00:07:19:11 Sometimes I refer these bacteria as Ashley and Carol.
00:07:22:16 And so you'll see the drag versions of the bacteria
00:07:25:11 throughout the talk.
00:07:27:10 But purifying an antibiotic
00:07:28:21 from an interaction is actually quite challenging.
00:07:31:11 So I found other Couchioplanes that we've isolated
00:07:34:18 from the soil and found that another strain T094
00:07:39:06 actually produces this antibiotic
00:07:41:09 on its own under certain nutrient conditions.
00:07:44:07 And I started working with Arthrobacter globiformis
00:07:47:03 a different indicator strain
00:07:48:15 because it's easier to work with in the lab.
00:07:50:06 It grows a little bit faster.
00:07:51:20 So that's the scenario that I'm gonna follow
00:07:54:09 up with for the rest of my talk.
00:07:56:07 So I showed you how I discovered this candidate
00:07:58:09 this potential antibiotic but at this point
00:08:00:19 we still don't know what it is.
00:08:01:22 So we have to do some chemistry experiments
00:08:04:14 to figure out the structure.
00:08:07:02 So I performed multiple analytical chemistry experiments.
00:08:11:03 One of them we collaborated with Yongle Du and Wenjun Zhang
00:08:14:12 to perform nuclear magnetic resonance or NMR.
00:08:17:04 And then in our lab we performed liquid chromatography
00:08:19:20 tandem mass spectrometry or LCMSMS.
00:08:22:15 And those two techniques combined gave us the structure
00:08:25:01 that you're seeing on the screen of Dynaplanin A.
00:08:28:11 This is the most abundant version
00:08:29:20 of the antibiotic that was produced
00:08:32:02 but actually there are 14 variations
00:08:34:08 of Dynaplanins that we detected
00:08:36:02 in all of our Couchioplanes samples which is wild.
00:08:39:11 So I'm only gonna focus on Dynaplanin A today
00:08:42:01 but there are other variations and many people usually ask
00:08:46:08 why did I name it Dynaplanin?
00:08:48:02 Well of course I named it after me
00:08:50:08 my drag persona Dyna Cockus Rose
00:08:52:22 plus the producing bacterium Couchioplanes
00:08:55:01 so Dyna planes Dynaplanin.
00:08:57:19 Lovely.
00:08:59:23 So I was really interested in figuring out how
00:09:02:08 the Couchioplanes bacterium produced
00:09:04:12 this Dynaplanin antibiotic.
00:09:07:12 And the first step is to figure
00:09:08:17 out what the building blocks are
00:09:10:17 and using stable isotope labeling
00:09:12:21 I found that Octanoic acid Phenylalanine and Arginine
00:09:16:11 are the three pieces that could put together
00:09:18:10 to make a Dynaplanin A molecule
00:09:21:07 but we still don't know how that process happens.
00:09:23:21 To do that we need to figure out the genes
00:09:25:12 and the enzymes necessary.
00:09:28:06 Unfortunately all prediction software that we
00:09:30:14 were trying to use did not work in our favor.
00:09:33:23 So we had to really study the structure of Dynaplanin A
00:09:37:07 and the differences between those building blocks to figure
00:09:40:04 out what genes were necessary.
00:09:42:16 And this chemical bond between Phenylalanine and Arginine
00:09:46:00 that I've highlighted in yellow
00:09:47:19 is the key chemical reaction that we needed
00:09:51:15 to figure out what these Dynaplanin genes are.
00:09:54:20 So it turns out that those two carbon atoms
00:09:57:06 are actually called alpha carbons
00:09:59:02 of both Phenylalanine and Arginine.
00:10:01:06 And bonding two alpha carbons of amino acids together
00:10:04:05 is very very uncommon.
00:10:06:07 So that really helped us narrow down our search.
00:10:08:21 And we also realized that the Carboxylic acid groups
00:10:12:08 of both of those needed to be removed
00:10:14:09 by some kind of enzymes maybe at that same step
00:10:17:04 or maybe separate steps.
00:10:18:23 So using this information we found the dynZ gene
00:10:23:10 which I've highlighted in pink.
00:10:25:20 And that was the key chemical reaction that forms
00:10:29:08 that chemical bond.
00:10:30:14 It's called a PLP-dependent aminotransferase.
00:10:33:01 I'm not gonna go into the details
00:10:34:04 of the other genes right now but if you wanna
00:10:35:23 pause the video you can read the table below.
00:10:39:19 So in bacteria most genes that are next to each other
00:10:42:23 on the genome tend to work in similar processes.
00:10:46:03 So we actually found four other genes that we think
00:10:48:10 are part of the Dynaplanin production pathway.
00:10:52:10 So we know these five genes are in Couchioplanes caeruleus
00:10:55:16 and Couchioplanes makes Dynaplanins.
00:10:58:12 So normally if we're doing genetics
00:10:59:22 we'd want to delete those genes
00:11:01:08 from Couchioplanes and then see that Couchioplanes
00:11:04:11 no longer makes the Dynaplanins.
00:11:06:06 That way we can relate Dynaplanin production to these genes.
00:11:10:11 Unfortunately that is not possible in Couchioplanes.
00:11:13:22 No researcher including myself has been able to do that.
00:11:16:11 That was a very humbling experience.
00:11:19:06 But what we can do is we can take a copy
00:11:22:04 of these genes and we can put it into a second bacterium.
00:11:25:14 In this case we chose Streptomyces coelicolor.
00:11:28:04 It's a very well studied Streptomycete
00:11:30:19 and it's a model organism in our lab.
00:11:33:16 So if we copy these genes we need to put them
00:11:35:16 onto a circular piece of DNA called a plasmid
00:11:38:08 in order to put them into Streptomyces.
00:11:41:17 So I like to think of plasmids as an instruction manual
00:11:45:10 and normally it's a blank instruction manual
00:11:47:20 just pages with no instructions.
00:11:49:18 And so if we give this empty plasmid to Streptomyces
00:11:53:00 she doesn't make Dynaplanins as expected.
00:11:55:19 Great.
00:11:57:10 But if we add those five Dynaplanin genes onto the plasmid
00:12:01:08 now Streptomyces has the instructions
00:12:03:05 to produce Dynaplanins and we confirmed all of this
00:12:05:20 through LCMS experiments.
00:12:07:15 So that was incredible but there are five genes here.
00:12:11:04 And at this point we actually don't know
00:12:12:19 if all five genes are necessary
00:12:14:11 or maybe just a subset of them are necessary.
00:12:16:22 So we deleted each of those five genes from the plasmid
00:12:20:00 essentially ripped a page out from the instruction manual
00:12:22:21 and asked can Streptomyces still make the Dynaplanins?
00:12:26:19 And what we found is that dynY and dynZ
00:12:28:16 the red and pink genes are absolutely necessary.
00:12:31:12 If you don't have those you don't get Dynaplanins.
00:12:34:08 DynV we think is necessary in Couchioplanes
00:12:37:06 but it actually was not necessary in Streptomyces.
00:12:40:00 And that's because streptomyces already has
00:12:43:01 another enzyme in its genome that can probably
00:12:45:17 perform the same chemical reaction.
00:12:47:08 Then dynW is what's called a hypothetical protein.
00:12:50:00 We have no idea what it's doing.
00:12:52:11 And dynX we think is actually regulating
00:12:55:04 this entire process.
00:12:56:21 So now that we know how Couchioplanes makes the Dynaplanins
00:13:00:09 my next question was how do Dynaplanins work?
00:13:03:13 What do they do to kill other bacteria?
00:13:06:22 So to do this we actually had to take
00:13:08:20 kind of a backwards approach.
00:13:10:23 So let me walk you through this process.
00:13:13:15 So on this Petri dish we have Arthrobacter globiformis
00:13:17:13 what's called the wild type
00:13:18:16 or unmutated version of the strain.
00:13:20:11 This is the starting one that we've been working with.
00:13:23:06 There's also Couchioplanes caeruleus
00:13:24:19 that orange colony in the center.
00:13:26:15 And we know that Couchioplanes is making Dynaplanins
00:13:29:10 because there's a zone of clearance
00:13:30:22 that I've outlined in blue.
00:13:32:12 But what you'll notice is that there's another colony
00:13:34:18 next to Couchioplanes and that's actually
00:13:36:19 an Arthrobacter colony that is a mutant
00:13:39:13 and is Dynaplanin resistant.
00:13:41:12 So if you remember from my introduction
00:13:44:14 Couchioplanes is making the Dynaplanins
00:13:47:02 antibiotics in its cells and secreting them outside.
00:13:50:03 So there's a high concentration next
00:13:51:18 to the colony and a low concentration away.
00:13:54:18 So when we add Arthrobacter there's some amount
00:13:57:12 of Dynaplanins necessary to kill Arthrobacter
00:14:00:09 and anything lower than that
00:14:02:02 Arthrobacter's fine to grow on.
00:14:04:06 But if we have a colony growing next to
00:14:07:00 or closer to Couchioplanes that must mean
00:14:10:23 this Arthrobacter can grow on a higher concentration
00:14:13:20 of Dynaplanins meaning it's resistant
00:14:16:02 and there must be some kind of mutations
00:14:17:21 that allow it to be resistant.
00:14:20:10 So the goal of this next part is to figure
00:14:22:20 out what those mutations are
00:14:24:06 because if we can find why these strains are resistant
00:14:28:06 maybe we can figure out what Dynaplanin
00:14:30:03 is acting on under normal conditions.
00:14:34:06 So first I wanted to quantify quite how resistant they are
00:14:37:14 so we can take that wild type Arthrobacter
00:14:40:07 again the unmutated version and measure
00:14:42:12 the diameter of its zone of clearance.
00:14:44:11 That gives us a baseline for how sensitive the Arthrobacter
00:14:48:07 is to Dynaplanins.
00:14:50:18 And then we can take these mutants
00:14:52:08 and perform the same process.
00:14:53:23 What we expect is that their zone sizes will be smaller
00:14:56:22 because a higher concentration
00:14:58:05 of Dynaplanin is needed to kill them.
00:15:00:06 So a smaller zone of clearance
00:15:01:23 means that cell is more resistant.
00:15:04:14 So I perform this experiment for eight mutants
00:15:07:20 of Arthrobacter labeled P through Y on that graph.
00:15:11:01 And all of them were significantly smaller zone sizes
00:15:14:00 meaning that they are significantly more resistant
00:15:16:04 to Dynaplanins compared to the wild type.
00:15:18:15 So that's awesome.
00:15:20:00 This is great news but this still
00:15:22:09 doesn't tell us anything about the mutations.
00:15:24:09 So we had to sequence all the DNA
00:15:26:13 of these nine strains and compare
00:15:29:12 the eight mutants to our wild type.
00:15:31:23 And we found that all eight mutants had a mutation
00:15:34:21 in a 2-oxo acid dehydrogenase subunit E2 gene.
00:15:39:19 What a mouthful.
00:15:41:08 So what the heck does this do?
00:15:44:14 Well in all cells very generally
00:15:46:21 they have to take up new nutrients from the environment.
00:15:50:03 They have to metabolize those nutrients with enzymes
00:15:52:14 and then they can get energy out of those nutrients.
00:15:55:01 And some of the enzymes that are key
00:15:57:03 to metabolizing nutrients are called
00:15:59:01 the 2-oxo acid dehydrogenases.
00:16:01:13 There are three in most living organisms
00:16:04:09 that can perform slightly different chemical reactions.
00:16:06:19 But the three are actually really hard to differentiate
00:16:09:11 between because they are so similar
00:16:12:02 at a DNA sequence and protein sequence level.
00:16:15:00 So we know that antibiotics tend to target
00:16:18:00 these key cell processes.
00:16:20:06 And so potentially an antibiotic could inhibit one
00:16:23:10 of these enzymes by blocking its function
00:16:26:08 meaning that the cell can't metabolize nutrients
00:16:28:19 and can't get enough energy
00:16:30:06 essentially starving it to death.
00:16:31:23 So my question was can Dynaplanin A
00:16:35:08 block a 2-oxo acid dehydrogenase?
00:16:38:02 Can that explain why these cells die?
00:16:41:04 So we chose to use Pyruvate dehydrogenase
00:16:43:18 one of the most well studied proteins
00:16:45:18 to answer this question.
00:16:47:13 So again this Pyruvate dehydrogenase enzyme
00:16:50:04 is inside Arthrobacter's cells.
00:16:53:08 It's also in Ashley's cells
00:16:55:11 but we're not gonna focus on Ashley quite right now.
00:16:58:00 So I used computer modeling
00:17:01:06 and made a three dimensional version of this protein
00:17:04:08 which is the green mesh.
00:17:06:01 It's called a space fill model
00:17:07:17 of this enzyme and we're zoomed in at the catalytic site
00:17:11:15 which is where this chemical reaction happens.
00:17:13:23 So inside the enzyme there are two substrates
00:17:16:23 Acetyl-lipoyllysine in gray and Coenzyme A in blue.
00:17:20:14 Those two enter this enzyme.
00:17:22:12 And the whole point is to take the Acetyl group
00:17:25:02 which I've outlined in pink
00:17:26:17 and transfer it from lipoyllysine onto CoA.
00:17:29:20 That's it.
00:17:30:17 The whole point of this enzyme and it's essential to life.
00:17:34:00 And if that happens normal metabolism occurs
00:17:38:03 and the cell can get energy.
00:17:40:09 So we wanted to know where could Dynaplanin bind in this?
00:17:44:16 And all of our results suggest that Dynaplanin A
00:17:47:18 actually binds right in the center
00:17:49:12 of this entire process potentially blocking the binding
00:17:53:11 of Acetyl-lipoyllysine and Coenzyme A
00:17:56:12 or at least blocking that transfer reaction from happening.
00:17:59:23 Regardless that means that this enzyme
00:18:02:17 is not functioning properly which means that the cells
00:18:06:08 cannot quite metabolize nutrients like they're supposed to
00:18:09:15 and they'll end up starving to death.
00:18:11:19 So recapping this entire process
00:18:14:08 I went out into the soil and I started studying
00:18:17:11 bacteria in their natural habitat.
00:18:20:02 Specifically I studied an interaction
00:18:22:14 between Couchioplanes and Actinoplanes on Petri dishes.
00:18:25:22 They're competing for nutrients and Couchioplanes responds
00:18:29:11 by taking Octinoacid Phenylalinine and Arginine
00:18:33:13 uses the five gene Dynaplanin cluster
00:18:36:21 and creates a Dynaplanin A molecule
00:18:39:21 which it then secretes from it cells
00:18:41:21 and it enters the Actinoplanes where it binds
00:18:44:12 to an enzyme called 2-oxo acid dehydrogenase
00:18:47:15 shutting down its metabolism of nutrients
00:18:50:12 and starving it to death.
00:18:52:07 (Dyna laughs)
00:18:53:03 Drama fierce work.
00:18:56:15 So this is just one example of the ways
00:18:59:05 that we can study the natural ecology of bacteria
00:19:03:03 and other microbes and find brand new molecules
00:19:05:20 from that process.
00:19:07:13 So I wanna thank you for listening.
00:19:09:06 I wanna thank all of my collaborators and support systems
00:19:12:01 and if you're interested in learning more
00:19:13:08 about this my work was recently published in mBio.
00:19:17:08 Thanks so much.

Videos in this Talk

Part 1: Dylan McClung, PhD Interview
Audience:
- Student
- Researcher
- Educators
- Educators of Adv. Undergrad / Grad
Part 2: A newly discovered antibiotic
Audience:
- Student
- Researcher
- Educators
- Educators of Adv. Undergrad / Grad

Speaker: Dylan McClung

Total Duration: 0:30:03

Recorded: May 2022

All Talks in Microbiology

Talk Overview

There’s a lot of drama in bacterial competition for nutrients. In this Share Your Research Talk, Dylan describes his thesis research on two soil bacteria that compete over nutrients. One of these bacteria combines amino acids and fatty acids to create a newly discovered antibiotic. Dylan discovered how this antibiotic attacks another type of bacteria, essentially starving it of nutrients. These findings have implications for understanding bacterial interactions and the production of anti-microbial agents.

Speaker Bio

Dylan McClung

Dylan McClung, PhD aka Dyna Cockus Rose is a microbiologist and drag queen science communicator who discovered a new antibiotic from soil bacteria competing for nutrients. Dylan recently graduated from the University of California, Berkeley, with a PhD in Microbiology. He plans to focus more on science communication (in and out of drag), especially emphasizing… Continue Reading

Credits

Director: Thi Nguyen
Producer: Shelley Carter
Videographer: Eric Kornblum
Editors: Jiefei Yuan
Original Music: Marcus Bagala
Graphics: Chris George, Maggie Hubbard

Part 1: Dylan McClung, PhD Interview

Part 2: A newly discovered antibiotic

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