The Second Week – Mass Murder of GB1

Today is the last day of my first full work week here at CMOP and I have killed a lot of bacteria.

 

I have also been learning a lot and feel like I’ve already made progress, if not in the way of research, in learning laboratory work. I’ve learned how to use pipetman^©, label everything, operate the autoclave, pour plates, inoculate cultures, record data and observations, spread bacteria using sterile glass beads, take photos of our results, and many other science/research things that will be necessary for my internship this summer. I have also learned a lot about my project and feel like I have a grasp on what I’m actually working on.

 

I have read two sizable articles related to two-component signal transduction, which is how some bacteria respond to changes in environmental factors. It consists of a histidine protein kinase (HK) and a response regulator (RR). The HK has a portion exposed to environmental changes in the periplasmic space of the cell, and through some mechanism responds to a change in environmental state by phosphorylating. The phosphorylated HK then interacts with the response regulator to change it from an inactive, to active state, changing the function of the response regulator by changing its molecular surface area. In most cases the response regulator is a DNA transcriber, and turning it “on” or “off” allows it to transcribe a specific gene or not. Unless it requires a sigma factor for transcription, which adds another necessary regulation step. But transcription can also be prevented by an anti-sigma factors too.

 

So, basically, it’s complicated. And I feel like I have a very basic understanding of it, which is kind of exciting and means I am learning.

 

I also read an interesting article on Deinococcus radiodurans, which is a unique bacteria that exhibits high resistance to gamma radiation. I read the article because we are considering testing GB1 against gamma radiation to see if manganese oxidation may protect it from damage. However, this article seems to explain that the Deinococcus radiodurans seem more resistant because they are better able to repair DNA damage after irradiation. They hypothesized in the article that accumulation of Mn²⁺ complexes (like GB1 might do) may reduce the amount of damage from gamma radiation because it locks up reactive oxygen species (ROS) formed during radiation. These ROS cause damage to the proteins which repair DNA after radiation, so by stopping damage to these proteins, it allows the cell to repair itself (Daly, 2009).

 

You are probably wondering what I actually did this week, since the title of this blog involves the murdering of bacteria, and all I’ve been talking about so far is how bacteria protect themselves. We (my mentor, Kati Geszvain, and I) focused on testing the ability for oxidizing and non-oxidizing Pseudomonas putida GB1 bacteria to survive hydrogen peroxide and UV light. Our hypothesis is that the bacteria forms the manganese oxides to act as a shield against unfavorable environmental factors. To test this hypothesis, we added varying concentrations of hydrogen peroxide to liquid cultures of oxidizing and non-oxidizing bacteria and let them grow for a day. I also plated the oxidizing and non-oxidizing bacteria each on an LB and lept (two different types of media) plate with little paper circles soaked in the same varying dilutions of hydrogen peroxide as the liquid cultures. After letting these grow for a day we found that 3% hydrogen peroxide was lethal to both types, and that there was little noticeable difference between oxidizing and non-oxidizing bacteria. But we performed it again, doing a few things a little differently, with pretty much the same results. So, the first few rounds of death were due to hydrogen peroxide.

 

The other environmental factor we tested was UV light. We performed our first test Monday, which was inconclusive. Of course, this was because we exposed the plate to UV, but did not think that we needed to take the lid off. Those bacteria were lucky. The next day we set up another experiment, after talking to a few different people, of both oxidizing and non-oxidizing bacteria on lept and LB plates and exposed them to twenty, forty, and sixty second of UV light with the Petri dish lid covering half of the plate to compare how it grew with and without UV exposure. After one day of growth we had much more conclusive results, and ironically, the Petri dish lids are excellent UV blockers. Go figure. The oxidizing bacteria had more growth in the area exposed to UV than the non-oxidizing bacteria, even all the way out to sixty seconds of exposure. This was kind of exciting! So we decided to test four other strains of bacteria to see if it really was the oxidation of manganese protecting those few lucky bacteria. All the other strains we tested were not oxidizing, and as we predicted, did not survive the UV light. (for this test we narrowed it down to only LB plates for 20 seconds because that is where we saw the most noticeable difference in our first test.) Today, we spread two more strains of bacteria, one oxidizing and one non-oxidizing along with all the other bacteria to test again their resistance to UV light. We will come in to see results on Sunday.

 

Another test we decided to perform on Thursday, is the effect of adding other sources of carbon that are more difficult to break down for energy. The two we used were “hickory mixed tanning” and “humic substances”. We added a little bit of each of these to an oxidizing and non-oxidizing GB1 liquid culture of the bacteria and let them grow overnight. The results were interesting. The normal wild type GB1 tends to aggregate when it oxidizes, and in the culture with added hickory mixed tanning, the bacteria aggregated into bigger clumps, but in the humic substances culture, the mixture was more homogenous and had less clumps. We think that it means that the carbon sources affected the bacteria’s ability to oxidize. To test this, we are going to try to isolate the solids from the solution to mass them and compare masses. I think it’s interesting to see how this might affect their growth and oxidation, and kind of unexpected.

 

So most of you, if any are still reading by now, are probably a bit confused by all of this. It’s a little bit technical, but a lot of fun. Basically, we tested this bacteria under different environmental stresses, comparing how it acted while oxidizing or not oxidizing. The reason we’re doing this is to determine why in the world this bacteria oxidizes manganese, so that hopefully we can get more funding (all of this research so far is to supplement a grant) to study how it oxidizes and try to utilize it for future benefits, like cleaning up the environment. I am an environmental engineer after all.

 

This week has been a valuable learning experience for me. I’ve asked a lot of questions, and learned a lot of answers. I’ve also performed many simple lab techniques over and over again to obtain some seemingly interesting results. And with the aid of hydrogen peroxide and UV light performed mass murder of Pseudomonas putida GB1.  And a few of its relatives too.

 

Daly, M.J. (2009). A New perspective on radiation resistance based on deinococcus radiodurans. Microbiology, 7, 237-44.