Update, 2009

Well, I hope this isn't a trend - updating my site only annually! Anyway, some nice news to relate. First, I have a manuscript recently published in the Nature ISME Journal with my friend and colleague Jamie Foster on the subject of cyanobacteria in marine stromatolites. Currently, this article is freely available to download. Also, Jen Blank and I have published an article on the alkaline spring system in the Del Puerto Ophiolite (described in an earlier post). This article is available as a corrected proof. Also, quite exciting, as part of the research with my new laboratory, we have just described a new species of Geobacter, name Geobacter daltonii FRC-32. This article is now in press in the International Journal of Systematic and Evolutionary Microbiology (IJSEM). Amazingly, G. daltonii and its nearest neighbor G. uraniireducens Rf4, are highly similar by 16S rRNA gene sequence (98.1% similar), but have divergent genomes (21% by DNA-DNA hybridization analysis). This should be published and available in the near future.

New Publication in the ISME Journal

Well, it seems like I've only been posting when I get a manuscript published. We'll see how long this system can keep up!

Anyway, the new manuscript is:

Green SJ, Blackford CA, Bucki P, Jahnke LL and Prufert-Bebout L. 2008. A salinity and sulfate manipulation of hypersaline microbial mats reveals stasis in the cyanobacterial community structure. ISME Journal 2:457-470

In this study we examined the effect of salinity and sulfate manipulations on cyanobacterial community composition and relative abundance in hypersaline microbial mats. To do so, we employed several cultivation-independent molecular analyses, including: cyanobacterial-specific PCR-DGGE analysis and colony-PCR with cyanobacterial primers. We demonstrate that the relative abundance of cyanobacteria, as assayed by PCR, was not significantly affected by the manipulations. We demonstrate how colony-PCR with specific population primers can be used to monitor relative abundance without heavy sequencing. Furthermore, the overall cyanobacterial community was only modestly impacted by the salinity and sulfate manipulations, and the cyanobacterial populations that developed under the lowered sulfate and salinity conditions were most closely affiliated with other hypersaline microbial mat cyanobacteria.

This article is a featured article in the current issue of the ISME Journal, and is therefore freely accessible. But please contact me if you cannot get a copy.

Cheers,
Stefan

New Position and New Publication

Well, it has been a while since I updated my blog. My apologies, but the last few months have been quite hectic. I have taken a new position in the Oceanography Department of Florida State University (Tallahassee, FL). I am working for Dr. Joel Kostka on a Department of Energy project exploring bioremediation (or bioimobilization) and natural attenuation of Uranium-contaminated sites.

A paper from the project I was working on at NASA has just been published online at Environmental Microbiology. If you are interested, see: Smith et al. 2008. Shifts in methanogen community structure and function associated with long-term manipulation of sulfate and salinity in a hypersaline microbial mat. Environmental Microbiology 10(2):386–394.
Hope you had a great new year.

Cheers,
Stefan

New Publication

I'm pleased to report that the final manuscript from my thesis has just been published online in the new "International Society for Microbial Ecology (ISME) Journal". This manuscript is the culmination of my doctoral research and describes how the rhizosphere effect can affect different microbial taxa differently.

The manuscript is now available in print. Enjoy!

Green, S.J., Michel Jr., F.C., Hadar, Y. and D. Minz. [2007]. Contrasting patterns of seed and root colonization by bacteria from the genus Chryseobacterium and from the family Oxalobacteraceae. ISME Journal. 1:291-299.

Kiva.org

I know this is off-topic, but I recently discovered this organization: www.kiva.org. It is an organization which allows you to extent credit via micro loans ($25 or greater) to individuals. It is not a charitable contribution, but rather a zero-interest loan to help people start or expand businesses. It is amazing to see how a little bit of money can go so far. I am not affiliated or paid by Kiva in anyway. I simply think it is a wonderful idea that appears to be implemented in a well-organized manner. Cheers, Stefan

New Position

My friend Dave chided me for not updating my blog frequently enough. I must admit it is true. It is hard to believe that it has already been over 3 months since I last updated it. Anyway, I now have a new position as a research scientist at the SETI Institute. I am still working at the NASA-Ames Research Center, however. I am currently working on a few different projects, including hypersaline microbial mats from Guerrero Negro, Baja, Mexico. The Exobiology Department at NASA-ARC is planning another trip to the salt production facility - Exportadora de Sal S.A. de C.V. - located in Guerrero Negro, Baja, California Sur, Mexico. This trip we're planning to study some of the higher salinity ponds (on the order of 150 ppt salinity) as opposed to the lower, but still hypersaline ponds of about 85 ppt.

I also have an ongoing project with Dr. Jen Blank of the SETI Institute to study the geology, chemistry and biology of ophiolite rocks found in certain locations in Northern California. Ophiolites are pieces of ocean crust that have been lifted up to the surface and exposed. When these rocks undergo weathering, a number of interesting abiotic chemical reactions can occur, and the water chemistry is highly modified. For example, in the system we are studying (see below), the pH of the water in local streams is roughly 9.0, and the water has high levels of carbonate and magnesium. In addition, we are finding unique microbial communities associated with these waters. More on this as the work progresses.
Cheers,
Stefan

Back to California

I have just recently returned to California after a semester as a visiting assistant professor in the Department of Civil Engineering and Geological Sciences at the University of Notre Dame. My wife insisted that among the first things that I do, I should update my blog. I spent the semester teaching Environmental Microbiology and a laboratory class entitled Molecular Tools for Environmental Microbial Ecology. Well, now that the semester is over I can say that I quite enjoyed it, but it was a lot of work. It looks like I will be getting a position at the SETI institute for the time being, and I am looking for a full time faculty position, or more funding to help me remain at SETI/NASA. Anyway, it is good to be back. More soon.
Cheers,
Stefan

Unifrac Update

I received an email from Micah Hamady at the University of Colorado. He informed me that there is now an online version of UniFrac available, and can be found at thefollowing website:

http://bmf.colorado.edu/unifrac/index.psp

In addition, there is a manuscript available:

http://www.biomedcentral.com/1471-2105/7/371

This should be a welcome improvement from having to install and operate Unifrac from Python.

Cheers,

Stefan

FIBR Conference and New Job

I just recently returned from a conference in Bozeman, Montanta. This conference was funded by the NSF Project - Frontiers in Integrative Biological Research, and is part of an ongoing research program at the University of Montana. This research program is involved in investigating microbial communities in hot spring environments in Yellowstone to ask the question - "Do Species Matter in Microbial Communities?"(http://landresources.montana.edu/FIBR/). The conference was excellent, and one of the most intriguing pieces of information I learned was related to the genome sequences of two relatively closely related cyanobacteria of the genus Synechococcus. Two environmental isolates of this genus, recovered from different temperatures along a thermal gradient of a Yellowstone hot springs, had their full genomes sequences. Amazingly, when these genomes were assembled and compared, they were found to have very little synteny (genes or sequences occurring in the same order on chromosomes of different species), despite having ribosomal RNA gene sequences nearly 97% identical. These genomes can be found at The Institute for Genome Research (TIGR) (http://cmr.tigr.org/tigr-scripts/CMR/CmrHomePage.cgi).

Other interesting news. I will be teaching two courses at the University of Notre Dame this fall as a visiting assistant professor in the Civil Engineering and Geological Sciences Department (CEGEOS). The two courses will be Environmental Microbiology, and a laboratory course entitled Molecular Techniques for Microbial Ecology.

Return from ASM

Just got back from the ASM meeting in Orlando, FL. The meeting was good - amazing things being done with full genome comparisons, plus microarray gene expression analyses. Anyway, just a few notes here:

1) A new software package called CLC that is definately worth investigating. [http://www.clcbio.com/]

2) A product called Perfect Match® PCR Enhancer from Statagene. Here is the manual.

See an article by De Milito et al. 1995. I also found this comment in a molecular biology group:

"[Perfect match is] Tetramethyl Ammonium Chloride. It costs about $30 for 500 gms from Fisher, and stinks to high heaven.I titrated it between 10 uM (micromolar) and 1 nanomolar leaving allother conditions the same."

This is not verified information.

Cheers,
Stefan

Update

Well, good news. Another manuscript of mine has been accepted for publication in the journal Applied and Environmental Microbiology. Wooh! I just sent the galley proofs back to the editor. It really looks great. I had a very positive experience with AEM - not just because my manuscript was accepted. The peer-review was excellent - great commentary by two reviewers and by the editor as well. After acceptance, the page proofs were generated very rapidly, and I'm happy to say that the manuscript has been published. Reference:

Stefan J. Green, Ehud Inbar, Frederick C. Michel, Jr., Yitzhak Hadar, and Dror Minz. 2006. Succession of Bacterial Communities during Early Plant Development: Transition from Seed to Root and Effect of Compost Amendment. Appl. Environ. Microbiol. 72:3975-3983.


Anyway, things are moving along here at NASA. We finally received all the various bits of our real-time PCR machine from Bio-Rad. Since I still have no reagents, I haven't really been able to check out if the thing is working or not. But it looks nice! Also, a third sulfate experiment has been begun and I've gathered some microbial mat samples at the initial time point (before removal of sulfate). I'm hoping to examine microbial communities at different depths during a diurnal cycle in the mat.

The summer is shaping up to be very busy. Meeting in Orlando next month (ASM) and Vienna in August (ISME). Plus, I am co-advising a student working on microbial communities associated with ophiolites. Should be interesting research. However, my position is very much in flux as my fellowship is expiring at the end of September this year. More on the state of affairs as it becomes clear.

A publication of interest: A computer program ("TreeClimber") to analyze microbial communities via analysis of phylogenetic trees. This seems similar to UniFrac, but I haven't examined it closely. However, the documentation seems better than UniFrac. Find the article here. Find the website here.

Finally, I note that NCBI has started a "Probe" database. Should be very useful.

Cheers,
Stefan

UniFrac Analysis

Well, just a note of some interesting articles regarding new statistical techniques to analyze sequence libraries in a manner akin to analyzing DGGE profiles:

Lozupone, C. and R. Knight. 2005. UniFrac: a New Phylogenetic Method for Comparing Microbial Communities. AEM: 71: 8228-35.

And an application of UniFrac:
Ley, R.E. et al. 2005. Obesity alters gut microbial ecology. PNAS. 102:11070-11075.

To use UniFrac, you will need to download and operate "Python" software.
You will also need to download a special "Numeric" module. Make sure you download the Numeric, not the NumPy, module.
Then you will need to download the .zip file of the unifrac program (http://bayes.colorado.edu/unifrac.zip).

The UniFrac program is not trivial to use if you haven't had any experience with Python or similar languages. I've emailed Dr. Lozupone and she has indicated that eventually a web-based UniFrac program will be available. However, not yet.

You will need phylogenetic trees in Newick tree format as the input data file for this program.

DGGE Help Page

I am announcing the release of my DGGE Help Guide. I have spent some time reviewing the questions that have accumulated on the YahooGroups DGGE page and decided to compile a guide to answer many of the questions that come up repeatedly. The guide is not meant to be definitive, but it is meant to be a resource for users to find the information that they do need. I also put in some of my own personal opinions regarding PCR and DGGE.

I hope that people find the guide useful. I will update the guide as time goes along, and I will be happy to receive additional information from anyone interested in providing it. If anyone feels that there are sections that they would like to write, update or correct, please contact me.

I have the guide on another blog: http://ddgehelp.blogspot.com/
The entire text can be had in word format or PDF format. However, I have found that the PDF format does not retain the hyperlinks. For the intended format, please use the word file.

Monthly Update

Well, it seems about time for a monthly update. I had thought I might do this more routinely, but I guess this is what we can expect! I have added a few tidbits to the SuPER PCR page. These include citations to two very nice manuscripts. One is a method similar to SuPER PCR but for RNA and the other is a clever mechanism to circumvent the problem of plastid DNA in environmental studies of plant roots.

Things here are going well. I am in the midst of a major sequencing effort relating to the "Sulfate II" experiment that was conducted at NASA. The idea was to take hypersaline microbial mats (See Bebout et al. 2005; Minz et al. 1999 and many others) and incubate them under controlled conditions at standard salinity (85 ppt), lowered salinity (35 ppt), at standard sulfate concentrations (~60 mM) and at low sulfate concentrations (< 1 mM). After letting these mats acclimate to these conditions, a suite of geochemical measurements were taken (oxygen/sulfide profiles, methane evolution, etc), and I have been working, together with Jason Smith (U of Florida), Mary Hogan (NASA/ARC), Brad Bebout (NASA/ARC) and Lee Bebout (NASA/ARC), on analyzing the methanogens and sulfate reducers in the system. The preliminary analyses of the methanogens are quite striking and indicate a dramatic shift in population as a result of the removal of sulfate. More on that when all the data are in. No sequence data yet on the sulfate reducers.

I have also been performing some phylogenetic analyses with the program Mr.Bayes (discussed in a prior posting). However, I am having problems with the technique - with regard to the branch lengths that the method is yielding. I have noticed that the branchlengths produced by MrBayes are significantly longer than those I get from other treeing methods such as Neighbor Joining or Maximum Likelihood. I have observed that the branch lengths are roughly 2x those of NJ, and 2x thes equence difference (as estimated by BLAST or other aligning method). I realize that MrBayes branch lengths are "expected changes per site" and not necessarily the same as NJ branch lengths. Still, the 2x factor seems excessive. If anybody has any thoughts on this I would love to hear them. Overall, the branching order yielded by Mr. Bayes seems quite consistent with other methods.

Well, more soon.

Contract Renewal

Well, good news here. My NRC contract was renewed for a second year. It's nice to be employed. However, since the NRC fellowship is essentially limited to two years, it is time to start looking for another position. I have applied for some grants as well, but it will be quite a while before I hear about the results of those applications.

Things are starting to get quite busy. I have a number of sequencing projects to support a large amount of geochemical data acquired in a long-term project here at Ames. The projects involved adding nutrients (ammonia,nitrate or phosphorus) to microbial mats maintained in the greenhouses here, and a second major experiment in which salinity and sulfate levels were modulated. So, we are examining major microbial functional groups: methanogens, sulfate-reducers, photosynthetic populations, methane oxidizers, and nitrogen-fixers. More updates on the results of these experiments soon.

Also, happy new years (shana tova) and happy birthday (to me).

GenomiPHI Update

I've done a little more work with the GenomiPHI kit. It is not a panacea for all that ails with PCR done directly on environmental DNA. I have had mixed results with different samples. Some samples with weak PCR do not fare well with the kit while others do. I have also determined that halving the recommended volumes does not help - you should use their recommendations.

On the plus side, I performed a denaturing gradient gel electrophoresis (DGGE) analysis of an environmental DNA extract directly and after pre-amplification with the genomi-PHI kit. The bacterial 16S ribosomal RNA gene profiles look identical before and after pre-amplification, suggesting that the pre-amplification is not biased.

GenomiPHI

Well, it's been a little while since I've updated. These things happen. I've been playing a bit with this full genome amplification kit called GenomiPHI (www.genomiphi.com). The kit contains a DNA polymerase (Phi29) and random primers. The polymerase is quite nice because it is less sensitive to environmental contaminants (humics, etc) than can be co-extracted with DNA. It works at low temperatures (30 deg C), does not require thermal cycling and is a proofreading enzyme.

The enzyme is useful because it makes copies (up to 5000x) of total genomic DNA. Apparently this is an unbiased reaction (I haven't tested this myself). I have so far employed this technique to enhance PCR amplification of the mcrA gene from methanogenic archae (see below). The kit is about $150 for 25 reactions - not cheap, but not out of reach.

In high sulfate environments such as marine microbial mats, methanogens do not compete well with sulfate-reducing bacteria and are generally not abundant. Thus, the detection of methanogens by PCR with mcrA gene primers can be difficult. I tried to circumvent this problem by performing a pre-amplification with the genomiPHI kit. I took several DNA extracts from high sulfate mats and diluted them either 1/10th or 1/100th. 1 ul of these dilutions were then added to the genomiPHI reaction with random primers and run overnight at 30C. It turned out that diluting the initial DNA sample was not a great idea. The first figure shows the DNA after genomiPHI amplification on a 0.8% agarose gel (first 7 lanes, left to right) and a standard DNA extract (lane 8).

No obvious difference between dilutions was observed after the overnight pre-amplification (the first three lanes are the same sample, undiluted, 1/10 and 1/100th). These samples were then subject to standard PCR with mcrA primers. The first seven samples (left to right) are the PCR yield from the reaction containing DNA pre-amplified with the genomiPHI kit. The next 3 are the same samples amplified without pre-amplification. The next 1 (strong yield) is a PCR yield from a low-sulfate mat sample generating high levels of methane. The final lane is the negative control that unfortunately has a slight bit of contamination. Anyway, sample #1 yielded strong PCR amplification when the undiluted DNA extract was subject to GenomiPHI pre-amplification. At a 1/10th dilution the PCR yield was weaker and at a 1/100th dilution, no PCR product was detected even after pre-amplification. The same sample gave only a weak PCR yield when amplified directly from the original DNA extract. A dilution of the DNA extract prior to the pre-amplification turned out to be a poor idea, and these samples did not amplify well (except for #16) even after GenomiPHI amplification. More work to be done, but still, it is interesting.

If interested, check out this article: Gonzalez et al. 2005. Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to process difficult to amplify samples and low copy number sequences from natural environments. Environmental Microbiology. 7(7):1024-1028.

16S rRNA Gene Sequence Aligner (Online)

Here's an online sequence aligner for 16S rRNA gene sequences that I heard about from Ruth Ley at the MBL workshop. It solves one of the problems with the ARB aligner - namely that the ends of the sequences are scattered all over the place and you have to manually adjust them.
The only downside is that this aligner aligns the sequences to other databases such as Phil Hugenholz's. Still, nice to avoid the problem with the ends.


http://greengenes2.lbl.gov/cgi-bin/nph-NAST_align.cgi

Website for SuPER PCR

I had an idea to create a website specifically for SuPER PCR users. So, I just appended another blog page to mine and it will hopefully serve as a node for people interested in the method and looking for advice and any updates. It can be found here: http://www.superpcr.blogspot.com.

Other than that, I am busy running phylogenetic analyses on some sequence data . I am trying out a new (well , for me) phylogenetic package call Mr. Bayes. We'll see how it goes - it is going to be running for a few days here!

SuPER PCR and Streptomyces Paper

Well, sorry I haven't updated the site in a while. I've been at a Molecular Evolution class in Woods Hole, MA. The main coursework of the class has just finished, and now I'm here for one more week to analyze some of my data from my Ph.D. work. Unfortunately, I'm a bit sick and have been so for a few days. Hopefully a good nights' sleep will take care of it.

Anyway, I'm very excited. My methodology paper with Dror was just published in the journal of Applied and Environmental Microbiology. It's a nice little technique that we developed a while ago and have been trying to get the word out. The method is called Suicide Polymerase Endonuclease Restriction (SuPER) and is meant to operate as a prior step to PCR and other molecular analyses. The method helps deal with a problem frequently found in environmental studies - the predominance of a single type of DNA that overwhelms the detection of other types of DNA.

Also, my paper with Ehud (now a Ph.D. student at the Technion in Haifa) was just published online in the journal Microbial Ecology. So, overall, a good publication week! This article is about the effects of compost amendment to soil on bacteria from the genus Streptomyces developing on plant roots of plants grown in such soils.