The ominous clouds in the sky and the cool breeze in the air can only mean one thing – Summer is gone and it’s time to stop slacking off. With that in mind, I thought I would update everyone on how my work on Microbacterium oxydans has progressed. Now that the bioinformatics work is more or less done, I’ve been putting all of the information together and trying to make it sound somewhat coherent. I am currently halfway to the 500 word limit that the Journal of Bacteriology has imposed for genome announcements, so the end is definitely in sight but more work still needs to be done. It’s going to be interesting managing classes, this paper, and another project I’m working on (more on that later) but I’m optimistic that the paper will be completed within the next week or two.
M. oxydans infothat I’ve written about so far:
- Microbacterium have been found in many human associated environments (blood, radioactive sites, food) and also act as plant pathogens in some cases. TDU was found in a toilet (thanks David!)
- The a5 pipeline produced 1,131,749 paired end reads with an average length of 133 base pairs, giving 80.358 fold coverage after error checking and quality control were performed. The N50 value of the genome is 1,056,891 bp (this is a weighted median, denoting the contig size in which 50% of all bases are contained in contigs that size or greater). I still need to include scaffold and contig length information, such as median and mean values
- The size of the genome is 3,746,321 bp comprised of 44 contigs in 8 scaffolds
- RAST was used to automatically predict the genes present in the TDU sequence. The default RAST gene model was used to make these predictions. The program predicted 3,667 coding regions and 357 subsystems. I will probably go more in depth into this area of the paper
- A phylogenetic tree of 16s sequences was created to confirm TDU as M. oxydans. I still need to include information about percent identity
Once this is all taken care of, I will be undertaking a new, independent project through the Provost’s Undergraduate Fellowship. My proposed project will deal with milk pasteurization in regards to cheese safety and quality. I will be creating cheeses using pasteurized cow’s milk and raw, unpasteurized cow’s milk and assessing the microbial content of the final products to see if there is a correlation between pasteurized milk and cheese safety. I will look for and attempt to identify and quantify known cheese pathogens, such as E. coli, Salmonella, and Listeria monocytogenes. The debate between raw milk and pasteurized milk has been ongoing, but with this study I can hopefully provide a little bit of insight into the controversy. Right now I’m writing my proposal (due in 3 weeks, yikes!) and doing hours of research to make sure I am prepared to undergo this project, but it’s still early and I have a lot to learn.
UPDATE (10/15) – So I was able to put some more time into the paper and it looks like I’ve gotten all of the information I need down. I’ll be sending it off to David so he can critique it
I finally found a computer with some internet access (just moved apartments and I have no internet or television for a week) so I decided to update everyone on the status of my organism Microbacterium oydans (TDU).
The library construction went very well, producing a genome with only 44 contigs contained within 8 scaffolds. The total length of the genome was 3,746,321 base pairs, which is similar to the lengths of other Microbacterium genomes (3,982,034 for M. testaceum, 3,952,501 for M. yannicii). I used the RAST genome annotation program to identify genes within my genome . The genome contained 355 subsystems and 3667 coding sequences according to the annotation pipeline.
Since this isn’t my computer I will have to end this post prematurely for now. Once I can get some more sustained internet time I will provide additional information about this organism
Our genomic library preparation kit finally came in (about a week late) but neither of the mentors are around to explain how to use it, so I thought I’d do some research and see what I could find on the Microbacterium genus. Unfortunately, most Google results are for the Mycobacterium genus which, while a very fascinating genus I’m sure, does not help me at all.
I finally came across a study performed in the mid 1960’s which analyzed some characteristics of the Microbacterium genus. The term Microbacterium was proposed in the early 20th century to identify a particular group of very small, gram-positive, rod-shaped bacteria that have been found in many dairy products. An important characteristic of Microbacterium species is that they are unusually heat resistant. The researchers conducted a number of tests to learn more about the physiological characteristics of the genus, based on 25 unique strains.
Particularly of interest to me is the following:
- These bacteria grow aerobically, but some strains can grow under anaerobic conditions. However, they divide much more slowly and lack pigmentation when grown anaerobically. Each strain in the study grew at a decent rate (typically around 3 days of incubation, but as much as 7 days were needed in some cases) when incubated in the 30-37 degree C range. Only 5 strains grew at 39 degrees and only 3 strains grew at 9 degrees.
- All strains grew in mediums with a pH of 6.8 and 7.5 . More acidic conditions (pH < 6) yielded almost no growth from the strains.
- Some members of the genus have the ability to reduce nitrates and liquefy casein, while nearly every strain could hydrolyze gelatin
- 5 strains were able to withstand 85 degree heat, and every strain was able to withstand at least 60 degree heat
- There is doubt as t0 where this genus should be placed in a phylogenetic tree. At the time of publication of this study, the most recent proposal was to place Microbacterium within the Corynebacterium. The author, however, dismissed this based on what he observed in terms of heat resistance and ability to be cultured, noting large differences between the two.
Here is a link to the paper
Micrococcus luteus wasn’t interesting enough to warrant further analysis, so I have picked another organism, TDU, to begin constructing a genomic library of. It appears to be within the Microbacterium genus, and shows identical similarity with the species oxydans. We actually isolated several Microbacterium colonies throughout this project from different sources, so I had a number of samples of which I could choose from to begin moving forward with.
A phylogeny of the different Microbacterium samples we isolated was built by David Coil, to help me visualize how similar the samples are to the published genome. There is one completed and published genome in the Microbacterium genus, for the species testaceum, so the goal of the tree was to help me pick from the most divergent organisms to minimize the chances of a duplicate publication of the same organism’s genome. This tree shows the comparative similarities of the Microbacterium species we found with the published Microbacterium testaceum genome recovered, with the most divergent organisms appearing to the left. UPDATE: Two outgroups have been added to further illustrate the degree of divergence
M. testaceum 16S sequence_alignment_tree
The three samples I picked as the best candidates were AV2, TDU, and TFU (TJU was a difficult and sloppy process to isolate, so I played it safe and avoided it altogether). AV2 had very, very low concentrations of DNA in the genomic preparation, so the sample was discarded. TDU and TFU both contained high levels of genomic DNA in their genomic preparations, so both were still equally viable as candidates. When I checked the glycerol stocks of both organisms on plates however, TFU appeared to have slight contamination (which is really bad, considering these stocks are our last resource for obtaining pure samples of these organisms). This confirmed TDU as the Microbacterium oxydans sample that I will begin working with to construct a library of.
Currently, the dilution streak of TDU is incubating at 37 degrees C, and tomorrow I will begin the process of confirming the glycerol stock and begin the tagmentation reactions for the genomic library.
I am a member of the Biological Undergraduate Scholars Program (BUSP), a science-enrichment program aimed at helping EOP students excel in their science courses and obtain research positions (thanks to them I obtained a position in Dr. Eisen’s lab, which had the added bonus of turning me into a blogger!). I recently gave a presentation to my program coordinator and fellow second-year BUSPers that summarized the methods the Eisen undergraduates have used to isolate, purify, and analyze the 16S gene of various bacteria. Dr. Eisen mentioned in my previous blog post that a summary of our work thus far would help our readers better understand our methodology and final goals, so I figured the presentation would be a good little placeholder until something more formal is written up
The Undergraduate Genomic Sequencing Project
I will admit that the slides are scarce on detailed information, and instead give very general descriptions. After all, I had to actually address the audience during my presentation and not just read off the slides!
Consider reading this first if you have no idea what we are doing
Last week I submitted 6 organisms to the UCD Sequencing Facility after several weeks of isolating and purifying their 16S genes. My initial submission of 5 organisms went somewhat smoothly, though something managed to sneak into two of my samples and mix the forward sequences I received (for each organism, we must submit enough for both a forward and a reverse reaction to be sequenced. We receive these sequences and align the complementary bases to generate a consensus sequence). I had really hoped this time would go much more smoothly than last, but unfortunately 4 of my 12 reactions, all forward reactions, were mixed and thus indecipherable.
I suspected the forward primer might have been contaminated at some point, and looking at my initial submission the only mixed reactions were forward reactions as well. Seems pretty clear to me that some little critter was able to find his way into our forward primer and rain on my alignment parade. I had hoped that I would have a larger list of organisms by this point, but here is what I have analyzed so far
- Staphylococcus pasteuri
- Enterobacter ?
- Bacillus amylolequefaciens
- Micrococcus luteus? (the most “interesting” organism I’ve found, based on GOLD results)
I brought in 5 new samples that I can hopefully begin culturing in the coming days, and in the near future I will resubmit the forward reactions that failed using a new batch of forward primer.