Posts by Manolito Torralba

Trapping Microbes 750 miles north of the Arctic Circle

About 1% of all microbes are “culturable” in the lab. They are some of the most stubborn organisms requiring special and specific nutrients as well as optimal temperatures and conditions. So, how do we get the “unculturables” to be “culturable”? We make bacteria “traps”, where we take media, sandwich them between a membrane that cannot be penetrated by bacteria and another membrane that can be penetrated by bacteria. Bacteria can grow and migrate toward the trap and essentially get stuck within the membranes and we can cultivate them afterwards. What about bacteria that don’t migrate? We can account for these bacteria by taking them out of the soil, placing them in media and putting them back into the environment using devices called diffusion chambers. These diffusion chambers consist of two membranes that are able to hold bacteria and media yet are porous enough for nutrients and ions. Using these techniques we have already cultivated more than 15 previously uncultivated species. For year two of this project we intend to cultivate at least 90 new species for whole genome shotgun sequencing. WGS data from such new species will be incredibly useful in determining which metabolic pathways are present to allow these organisms to survive in such a harsh barren climate.

Diffusion Chambers

Diffusion Chambers

Setting Bacterial Traps

Setting Bacterial Traps

Traps Under Water

Traps Under Water

Thule, Greenland Year Two

Sequence data from the previous year allowed us to determine the overall microbial population in each site and this year we decided to focus on the Rich Lake site which seem to have representation of nearly all microbes found in the other sites. So lucky for us we only had to work on one site this year rather than six. This in itself had me excited to go back to Thule. After a five-hour flight on a military plane from BWI I finally arrived to Thule Greenland where we were greeted by the Colonel as well as other high ranking military officials at the hanger. Once I cleared the customs processing area, I arrived to the dorm where the other scientists were living. It was a little different from last year’s accommodations but nevertheless the luxuries of WI-FI, Internet and cable TV were all available. As I am anxious to get to the field and see the changes in the Rich Lake site, we were given some interesting news. That day was not a good day to travel to the site because a mother polar bear and her two cubs were spotted nearby not too long ago by military police. However, we managed to get other work done by preparing the schedule for the sampling, cultivation and other labwork.

 

The next few days consisted of preparing culture media, cultivation traps and diffusion chambers, and going out into the field (polar bear spray in hand; yes it’s a real thing!). We were extra careful in the field since there was quite a bit of fog in the area that did not seem to go anywhere and fog happens to be the same color as polar bears. The fog did however make it a bit easier to sleep since most of the sunlight was covered and when there’s 24 hours of daylight from mid-April until September, a little fog can still serve a purpose.

Rich Lake Site

Rich Lake Site

Greenland

Greenland

Thule, Greenland – Day One

Arrived at Thule, Greenland after a 5 hr flight from Copenhagen.  It was pretty interesting seeing a long line of people all getting on a flight that was headed to a part of the world that usually has less than 600 people there at any given time.  Arrival was pretty straightforward, no jetway, no customs, no LCD screens telling you where to pick up your bag.  Just a few military personnel checking your documents to ensure that you have the approval from the Danish government and USAF to be on base.  First impression getting off the plane…it’s cold.  Not as cold as I expected it to be but it was just 90 degrees F when I left home a few days ago.  Today’s high was 39 degrees F.  Standing in the sun it’s not so bad but when the wind starts blowing it turns into a recipe for chapped lips and windburn.  Oh and did I mention the massive mosquitos here?  Not much wildlife in this part of the world but the mosquitos outnumbers the vertebrates probably a million to one.  They are also VERY aggressive; they even swarmed the trucks while we were driving around the base.  We were shown our living quarters, which were very nice, kind of reminded me of living in the dorms during undergrad.  There are individual rooms and a shared bathroom on each floor.  We toured the various sites that our collaborator Slava Epstein already pointed out as good sampling sites that vary in vegetation and proximity to water.  The land here is quite desolate, not much green, mostly moss and small shrubs growing.  Traditional trees are nonexistent but “ground trees” are actually common.  They are trees that grow outward on the grass and not upward.  The rest resembles pictures taken by the mars rover.  As the day goes by I noticed the sun was circling and I came to the realization that the typical artic summer was happening right in front of me.  The sun literally circles and will not go down until around September.  It was quite odd, getting in bed at midnight and seeing the sun still in the sky.  Tomorrow will be more interesting since we will be going further away from base to sample additional areas. 

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blog1

Thule, Greenland – Day Three

Day three started with me missing breakfast. It seems that folks around here only eat breakfast between 5am and 8am. Today was a very rough day for sampling.  About an hour drive to the area near the site, about a three-mile hike to one spot another half-mile hike to another spot followed by the three and a half mile hike back to the truck. We sampled “rich” soil and “rich” soil from a lake. These two sites were sampled and categorized as “rich” due to the abundance of vegetation around and near the sites. The area surrounding Thule is very desolate so I can imagine the plants have a hard enough time growing.  It would be very interesting to see what microbes are present in these two sites to allow such vegetation to grow; even more interesting to see how water affects the microbial population. Samples were frozen once we got back to the on site lab. A small portion was saturated with AllProtect to ensure preservation of RNA for transcriptomics analysis.

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The day ended with a lecture from another NSF grant recipient to install a telescope on the Greenlandic ice cap. It was an interesting idea to coordinate radio imaging from other telescopes around the world to look at quantum singularities that were very far away. After speaking to some of the other scientists here I found out that our group, which includes myself and our collaborators Slava Epstein and Dawoon Jung, were the ONLY Microbiologists on the base. Everyone else was either a Geologist, Environmental Scientist, Astronomer, or Meteorologist. It was great to hear about everyone else’s projects.

The Microbiome of Esophageal Cancer

In anticipation of the International Human Microbiome Congress, our group has diligently worked to generate data to present for our HMP demo project studying the microbiome of patients who have developed esophageal cancer, gastrointestinal reflux disease, and barrett’s esophagus.  We received a large number of samples in December of 2010 which surveyed four body sites (esophagus, fecal, oral and stomach) of twelve patients.  Upon isolation of DNA, we amplified a variable region of the 16S gene for each sample using barcoded PCR primers.  Incorporation of the 454 A and B adaptors to our primers also provided minimal loss of sequence data when compared to previous methods that would ligate the adaptors to amplicons after PCR.  This method also allowed us to generate sequence reads which are all in the same 5’-3’ orientation.  A large dataset with high quality sequence reads was generated and is currently going thru phylogenetic analysis.  Metagenomic data is also currently being generated from DNA extracted from esophageal brushings taken from a healthy individual as well as a patient who has developed esophageal cancer.  This comparative analysis will be scientifically beneficial in identifying key structural and functional elements that are known to increase pathogenesis of a complex disease such as cancer.  We are anxiously awaiting results from the analysis of these sequences and expect to present a thorough investigation on the esophagus microbiome.

HMP Consortium – St. Louis Missouri

Human Microbiome Project Consortium – September 2010 – St Louis, Missouri

We received warm welcome messages from Dr George Weinstock and Dr Jane Petersen as well as a humorous welcome from Dr Larry Shapiro, Dean of Washington University Medical School. 

It was wonderful to see so many scientists come together to share the progress on their individual HMP related demonstration projects.  Our own demonstration project with Dr Zhiheng Pei, involving the esophagus microbiome and how that relates to esophageal adenocarcinoma (EA), was quite unique compared to the other projects as we were the only group to focus on the correlation between bacterial population and a form of cancer. 

With over 400 participants and 59 speakers, the conference was quite successful and very interesting.  JCVI Director Dr Karen Nelson did a wonderful job moderating one of the segments.  Dr Roger Lasken also gave a thorough presentation on his lab’s single cell approaches to genomic sequencing of uncultureable bacteria.  Johannes Goll gave a great presentation on his recent work with an open source tool called METAREP (recently published in Bioinformatics 8/26/2010), which is designed to help scientists with analyzing annotated metagenomic data.  And Dan Haft presented his interesting work with algorithmically tuning protein families from reference genomes for systems discovery. 

Overall the conference was quite interesting and informative.  I continue to wish all of the participating sequencing centers, PIs, and others involved with the HMP much success with their projects. 

Hope to see everyone in Vancouver!!!