Planetary Protection (Reverse Contamination)

This is the third of three posts about the planetary protection workshop I attended at NASA Ames from March 24-26, 2015. The first is here.

I mentioned, in my last post on forward contamination, that reverse contamination is the primary concern for Planetary Protection (PP). In this context, reverse contamination refers to the transport of Martian life to Earth. Although, I did hear a totally different definition from Jen Law (the current flight surgeon for the ISS,) which I will get back to later.

I totally get why the primary mission of Planetary Protection is to, well, you know, protect OUR planet. After all, life on Earth has a very long history of ensuring the survival of life on Earth. But, from my perspective, it seems like less of a concern than forward contamination, which all seem to agree is inevitable. It’s tempting, as someone whose job is NOT to contribute to policy on this topic, to roll my eyes when people in the room suggest that astronauts returning from a Martian collecting trip might show signs of illness due to infection by a Martian microbe. First of all, see previous post for the extreme survival skills required to get by on Mars and then ask yourself: what are the odds that something adapted to life on Mars is likely to become a human pathogen upon first contact? Not very? Really, no one has idea. But, let’s assume that a Martian microbe actually has infected a human astronaut, and we have a sick astronaut on a long flight home, then ask: how would we attribute the illness to the Martian microbe? Are we going to apply Koch’s Postulates during the return voyage? Are we going to do some high-throughput 16S rDNA surveys or metagenomics? If we do, will we find evidence of familiar human pathogens. Of course we will. Wait, do Martian microbes even have DNA? It must be the case that other Planetary Protection workshops will be addressing these issues, because every time I brought them up people were like, “Yeah, yeah, we know that stuff is important and difficult.” But, no talks on these subjects at this workshop. Maybe next time!

So, with respect to avoiding reverse contamination, the main objective is to, as they say, “break the chain” (BTC) of contact with Mars. Bob Gershman discussed the technology under development to meet this objective for a robotic Martian sampling trip. According to the NASA procedural requirements (see NPR 8020.12), “Samples returned from Mars by spacecraft should be contained and treated as though potentially hazardous until proven otherwise.” How contained is contained? The PP Office has a “draft” requirement of < 0.000001 probability of inadvertent release of a single unsterilized Mars particle to the Earth’s biosphere. I MEAN. How the hell do you make that calculation!? What’s really cool is that they will actually figure that out.

So, anyway, there needs to be the sealing of compartments and the external sterilization of components and the withstanding of very unpleasant Earth re-entry conditions, including extreme heat and force. Gershman presented a very detailed, technical account of various sealants and containment materials and reentry technological developments. I was relying pretty heavily on my audio recorder for this talk, because it was too information-dense for me to take useful notes, but it died. So, I’ll share with you an excerpt from his abstract.

“Sealing modalities being investigated include brazing, explosive welding, bagging, and conventional o-rings. Sterilization modalities include heat, pyrotechnic paint, plasma, and hydrogen peroxide; but it should be noted that NASA has not yet considered which of these – if any – could be certified for sterilizing Mars material. Also, technology is needed to assure (with an unprecedented degree of confidence) that the Earth entry vehicle would withstand the thermal and structural rigors of Earth atmosphere entry and that the sample container and its seals would survive Earth entry, descent, and landing. Concepts for a new Earth entry vehicle that could satisfy the stringent MSR reliability requirements have been under study for several years, including some preliminary technology development activities.”

So, basically, we’ve thought a lot more about forward contamination because that draws upon our understanding of sterilization and contamination in the context of human health and microbial monitoring, a la CDC, DHS, DOD, etc. The procedures and technologies for dealing with safely bringing Marian life to Earth are very much under development.

I mentioned that the flight surgeon in the room was using a different definition for reverse contamination. She was referring to the fact that, once the astronauts return to Earth, they are likely to have compromised immune systems and altered microbiomes. They may be at risk from exposure to common Earth microbes. I thought this was an interesting take on “reverse contamination.” Also, it brings up the topic of microbiomes, which is why I was in the room. I was there to talk about the microbiology of the built environment and share some of the results from Project MERCCURI’s microbial ecology of the International Space Station. The ISS represents an extreme built environment for a number of reasons, microgravity, high radiation levels, very little exposure to Earth air, very little flux of Earth microbes. The spacecraft that carries humans to Mars will experience extremes of those extremes. How will the microbiomes of the astronauts and their homes respond? Don’t worry, NASA is on that, too.

What the fungi do I do with my ITS library (Part 2)

Previously, I expressed some concern about size variation in my environmental fungal ITS PCR libraries. I’m still concerned about that, but I have an additional concern. The ITS region can’t be aligned, and I’m partial to phylogenetic approaches to pretty much everything. So maybe ITS is not for me?

So, I asked Twitter again…

[View the story “What the fungi do I do with my ITS library? (Pt. 2)” on Storify]

In summary, I don’t think that I can use ITS given the size variation that I see, and I’m not sure that I want to, given the fact that you cannot align it to do phylogeny-based analyses.

28S (or LSU) is a reasonable alternative to ITS that has two big downsides: 1) the reference database is much smaller than the ITS reference database and 2) it does not provide the fine-scale taxonomic resolution that ITS does.

Rachel Adams referred me to Amend et al,  in which they use both. I’ll have to look into this approach…

What the fungi do I do with my ITS library?

It’s been about 8 years since I started working on my first 16S rRNA PCR survey (of Drosophila gut microbes). At that time, I was occasionally asked, “what about Archaea or what about microbial Eukayrotes?” Then, and ever since, my reply has been that it’s hard enough to get a handle on what’s going on with the bacteria – I don’t need to make my life more challenging by broadening my scope.

But, finally, this month, I’m making my life more challenging. As part of my new Seagrass Microbiome Project, I’ve decided to tackle the fungi. As far as I can tell, ITS is the “barcoding” marker of choice for fungal types. For many reasons, it’s best to follow the herd when doing this sort of thing: 1) someone else has already designed, tested, and published results with these primers, 2) there is a reasonably large database of ITS sequences available to compare my sequences to, and 3) I lack the interest and personnel to explore an alternative approach.

So, I just plunged right in. At first, I tried some new primers designed by Nick Bokulich, but he warned me that they were “finicky” and he was correct. I got no amplification with my seagrass samples, and the positive control I had only worked about half the time. I know some other fungi people, well, I know Jason Stajich (@hyphaltip), so I asked him which primers I should use, and I decided to go with the primers set used in a cool paper by Noah Fierer’s lab, in which they looked at fungi in rooftop gardens in New York City.

Those worked, and a few days ago I got word that my sequencing run was in. It looks like crap. We typically get about 12 million sequences from our MiSeq runs, but this time, I only got 4 million. I was also told that the reverse reads looked much better than the forward reads.

So, now, in addition to working with a new “barcode,” I have to troubleshoot a crappy sequencing run. In many ways, it’s nice to have undergrads and a technician in the lab who do all of my lab work for me these days, but it sucks when it’s time to troubleshoot because I’m so far removed from the bench that I have no idea what’s going on anymore.

So, the first thing I asked for was the Bioanalyzer trace that’s always run before the library goes on the machine. It looks like this:

Bioanalyzer trace for my first fungal ITS MiSeq run

Bioanalyzer trace for my first fungal ITS MiSeq run

I had been told that there was size variation. I had even seen some of the PCR gels. But, still this is not what I expected to see. Upon seeing this, I am concerned about two things. 1) If there is strong preferential amplification of smaller DNA molecules during the bridge PCR on the flow cell, then will I even see DNA from those larger peaks? 2) With our 300bp reads, for sure the amplicons in the peaks <400 will have overlapping forward and reverse reads, but for sure the 676bp amplicons will not. What effect will these two things have on my analysis? How do I accommodate this size variation? One of the reasons to follow the herd with these methods is that other people have probably already encountered and dealt with exactly this issue, so I turned to Twitter…

There are some great resources suggested here. I know what I’ll be reading this weekend…

MicroBioBlitz

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As soon as I was introduced to the concept of a BioBlitz, I immediately emailed the boss man Jonathan Eisen saying that we should arm these people with microbial sampling kits. He, apparently having received the same BioBlitz announcement that I did, was seconds away from sending the same email to me!

We contacted Jack Gilbert at the Earth Microbiome Project and Janet Jansson at LBL/JGI/JBEI, and George Phillips, the organizer of the Mount Diablo BioBlitz, and they were all on board to help make a MicroBioBlitz happen.

A few days ago, I received another announcement about the 2nd annual UC Davis SEEDS BioBlitz. So, yesterday, I got the go-ahead from those organizers, last night I threw together 20 swab kits, and today I took them to the Putah Creek Riparian Reserve for a test run of the MicroBioBlitz concept. It was So. Much. Fun! The BioBlitzers were super-excited to start swabbing their organisms of choice. I gave them no guidance, and I can’t wait to see what they decided to swab! Before I left, they had swabbed lizard butts and oak galls, and they were on the lookout for otter scat!

I couldn’t stay for the whole event because Vivian needed a lunch and a nap, but before we left, I had her swab a Valley Oak for us! She loved helping, but cried when I took the swab away from her, so I gave her a swab to play with, and she spent the next 20 minutes swabbing everything she could reach!

MBL #3 – The Volta Experiment

Before you even have a chance to really get to know your classmates, you are led on a dark walk to a stinky Cedar Pond, where the brave venture chest-deep into the cold, sulfuric muck. They stomp around in the soft, slimy, decomposing leaf litter and sediment, releasing bubbles of methane gas. The methane is trapped by one person (Dan Buckley, in this video) in a stopped funnel. On the count of three, Dan removes the stopper and Steve Zinder holds a flame as the methane shoots out of the funnel. Voila! Volta!

MBL # 2 – Jorg Overmann and my pet phototrophs

I mentioned in my last post that one of the many wonderful things about the Microbial Diversity Course at the MBL is the quality of the invited speakers. Another favorite of mine was Jorg Overmann, who wrote the book on Molecular Symbioses and is the director of the DSMZ (I did not know that!) He introduced us to his favorite bugs, the consortium known as Chlorochromatium aggregatum.

A really nice summary of what we know about them is here on the Small Things Considered blog. Basically, there is a flagellated Beta-Proteobacterial rod that is encrusted with non-motile green sulfur photosynthetic symbionts (epibionts, to be precise, because they are attached to the surface.) Their relationship is complex, but what’s overtly cool about it is that the rod can chauffeur the epibionts around in search of the right conditions for photosynthesis. One can (and we did) see their cool scotophobic behavior in a light microscope. It looks like this (run towards the light!):

Source

Jorg said that I should be able to find these consortia in the nearby Trunk River, so hello, of course I will try to enrich for them! However, he strongly discouraged my reliance on their enrichment for my independent research project in the last half of the course, because they are quite elusive and grow very slowly.

So, let me backup a little here. The first three weeks of the course you attend lectures in the morning and then you are in the lab until late at night. Those weeks are spent attempting to culture a diversity of isolates from environmental samples that you collected in the first few days. You are also introduced to all of the super-cool equipment housed at the lab, to which you will have full access. For the last three weeks of the course, you draw upon your new-found knowledge, the support of those around you (students, instructors, TAs), and the abundant resources at the MBL to work on an independent research project. Most people work on AN independent research project, but I apparently am not most people. I worked on FOUR. This is the first (and favorite) that I will describe in a series of blog posts.

Setting up the enrichment is easy. You just take some sediment from the river, put it in a bottle with some spring water supplemented with sodium sulfide, making sure that there is no head space and no way for oxygen to get in. Then, incubate it at room temperature with a light-dark cycle and wait.

Then you wait. I waited for 23 days. Some of them had purplish top layers of sediment and some had greenish top layers. Jorg said that Chlorochromatium aggregatum will form a biofilm on the surface of the glass. A biofilm formed! On the surface of the glass! It was purple.

Then you play with your biofilm. First of all, before I proceed, I did NOT find any consortia, but I hope you will not be too disappointed, because what the hell was I going to do with them in the week that I had left anyway? My biofilm consisted primarily of two cell types (see below.) You can see big, oval cells that contain highly refractive sulfur granules, and you can see some smaller, dark rod-shaped things.

I decided to use one of our cool toys to see if I could physically isolate the presumed purple sulfur bacteria (encircled in yellow) from those other guys. I wanted to isolate them 1) to get a 16S PCR product to identify them and 2) to see if I could transfer them from the biofilm into new culture medium and grow them.

I used the PALM CombiSystem which is a laser dissection/optical tweezer type microscope. Basically, I could spread out my biofilm on a slide covered by a very thin membrane, then use a laser to cut out a circle around one or more cells on the membrane, and then use some mysterious quantum force to catapult the membrane+cell(s) directly into the cap of an Eppendorf tube (filled with culture medium or whatever.) What the… what!?!? That was supercrazycool! Somewhere I have a movie of it, but you can check this out to get an idea:

So, I was able to isolate these guys (they were Chromatiaceae, per RDP) for 16S PCR and I was able to get them to survive the transit into the Eppendorf tube. After two days, they were still metabolically active!

These figures are from my final report. I must admit that I wrote the bulk of it, while drinking beer, and during an all-nighter. So, I wont vouch for it’s quality and can’t recommend that you read it. These reports, regardless of the quality, are good to have around because current students pore over old reports for inspiration. Reading it myself after 3 years, I felt a strange combination of “Wow, I did that?” and “Eww, I wrote that?” But, my goal here is just to share a little bit of my MBL experience with the world, and I hope you enjoy it.

If you were at Lake Arrowhead, you should know the MBL

The recent Lake Arrowhead Microbial Genomics Conference (#LAMG12) was the first time I’ve been reunited with one of my classmates, Ben Tully, (@phantomBugs) from the MBL. (In June 2009, I spent 6 weeks at the Microbial Diversity Course at the Marine Biological Laboratory in Woods Hole.) On several occasions, we explained to people at the conference how we met, and were met with blank stares.

Just as Laura Sauder (@LauraASauder) forgave the medical microbiology folks in the room for not recognizing a photo of Sergei Winogradsky, I am willing to do the same for those who do not recognize the Microbial Diversity course at the MBL. But, if your flavor of microbiology has anything to do with the words environment, community, genomics, metagenomics, evolution, or ecology, then you should fall into one of three categories: 1) You went to the course. 2) You are going to the course. 3) You want to go to the course.

Here is some text from the course website:

Students will isolate, cultivate, and experiment with characteristic microbial types from various marine, freshwater and terrestrial habitats, including those microbes living symbiotically with animals and plants. Emphasis will be on the isolation and cultivation of organisms that are distinguished by their phylogenetic, physiological, and morphological properties. Techniques for cultivation of strict anaerobes and phototrophs will be emphasized. Examples of microbial types that will be isolated are methanogens, acetogens, sulfate-reducing anaerobes, fermentative anaerobes and both oxygenic and anoxygenic phototrophs, as well as bacteria involved in the geochemical cycling of various metals. Magnetic bacteria, sulfur-oxidizing bacteria, spirochetes, and luminescent bacteria will also be studied. A laboratory component on molecular approaches to microbial diversity will instruct students to use approaches of molecular phylogeny and comparative genomics. This will involve isolation and amplification of 16S rRNA genes as phylogenetic markers and the use of computer software programs to analyze nucleic acid sequences and to construct phylogenetic trees. As the capstone activity of the course, participants will conduct an individual research project of their own design.

You will learn a LOT about microbes and how they do what they do. You will become a microbiologist. The way you view the world will be permanently altered. Just ask anyone who has attended the course. But, what that blurb does not tell you is that every day, you sit through lectures or talks by the world’s expert on a given subject. Time to learn about thermophiles? Here is Karl Stetter. And, not only will he talk to you in the classroom about his research, he will stick around for a few days. He will hang out in the lab, eat meals with you, and he will be part of the crowd that makes the trek to the secret beach when the bar closes.

It’s just a really, really special place, and if you were at Lake Arrowhead, if you were at ASM, if you were at ISME, (I know there are more, but you get the idea) you must fall into one of those three categories.

More MBL posts to come…