Comparing the effects of arabinate and glucose in compensating for growth defects in mutant strains of H. salinarumAbhishek Kulgod, Senita Portlock, Chase Yuan, Suzanne Zhou, and Xueqi ZhuNorth Carolina School of Science and Mathematics

IntroductionIn extreme conditions, certain microorganisms have evolved unique methods necessary to adapt to high salt, pH, or temperature conditions. Of these “extremophiles”, those in the Archaea domain of life have immense implications for biotechnology. Understanding how these microorganisms adapt to extreme conditions can help humans apply the same techniques in industry to develop genetically engineered products. Many archaea have the potential for significant biotechnological advantages, but have not yet been fully developed.

Current LiteratureGlucose can offset defects arising from deletion of transcription factor trmB, which is associated with metabolic pathways in Halobacterium salinarium (Schmid et al., 2009).

Arabinate, a derivative of glucose, has not yet been tested with respect to cell growth.

Studying arabinate can help specify the function of trmB and its corresponding metabolic gene network while also developing use of the sugar in future studies of halobacteria.

HypothesesDoes introducing various concentrations of glucose compensate for the known growth deficiency in the Δtrmb strain?

H0: μ trmb/glu = μ trmb

H1: μ trmb/glu > μ trmb

As a glucose derivative, does arabinate reproduce the growth trends found in the Δtrmb strain with glucose?

H0: μ trmb/glu ≠ μ trmb/arab

H2: μ trmb/glu = μ trmb/arab

Serial Dilution of Sugars

*Note: After adding the sugar mixture to the cells at a 1:1 ratio, the above dilutions were halved

MethodsH. salinarum and a trmB knockout strain were collected

Both strains were diluted in growth media and treated with 5 concentrations of glucose or arabinate.

We performed a 1:5 serial dilution from 10% and added a control group.

Following 1.5 days of incubation with the optical densities analyzed every 30 minutes, the archaea and data were collected.

The following graphs display the logistically fitted growth rates of H. salinarum at four concentrations of arabinate or glucose solution and a sugarless control.

Figure A: Serves to demonstrate that the wild type strain of the archaea is not as affected by the addition of sugar as the trmB-deficient mutant. Demonstrated by the small difference between the lines steady states.

Figure B: Demonstrates that adding glucose will still affect the growth rate of the archaea, similar to the way archaea responded to arabinate. Could be a result of lab error and requires further examination.

Figure C: Suggests that 1% arabinate is the optimum concentration for maximizing growth rate in the absence of the gene trmB.

Figure D: Introduction of glucose into mutant H. salinarum compensates for the lack of trmB. Limit to the amount of glucose that can be added beneficially, between concentrations of 0.20% and 0.04%.

Figure E: Compares the growth rates of the mutant strain ΔtrmB of H. salinarumin 5% sugar concentration with both ΔtrmB in sugarless control and Δura3 in 0% sugar. Simultaneously demonstrates the effect of adding sugar to ΔtrmB to induce increased growth rate and compares that result with the wild type still with the trmB gene.

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Quick ResultsWhile testing different solution concentrations with each strain of archaea, we found a correlation between increased growth rate and increased sugar concentration.

The control group of ΔtrmB had a maximum growth lower than that of the wild type control group.

Key Points from FiguresWhen glucose is added to the ΔtrmB mutant, the growth rate increases (Fig. D).

Similarly, with arabinate, the original growth rate of the mutant archaea started out lower than the wild type (Fig. E).

With the addition of 5% arabinate solution, there was a 29.4% increase in the growth rate of ΔtrmB and a 28.6% increase in the growth rate of Δura3.

While in previous glucose studies was found to have a negligible impact on growth of wild type archaea (Schmid et.al., 2009), we found that arabinate improved growth in the wild type (Fig. E ).

Further ResearchIt is possible to test the growth of H. salinarum in other different types of sugar, such as ribose, to see if it responds in a similar manner

We can fine tune the optimal sugar concentration by performing smaller iterations in the series. Research into which sugar improve H. salinarum growth furthers our knowledge about the metabolic pathways of them and their potential uses.

Acknowledgements and CitationsThanks to the following individuals for contributing to our project:

Allison Edgar

Jordan Gulli

Paul Magwene

Colin Maxwell

Amy Schmid

Rotem Ben-Shachar

Amy Sheck

Horia Todor

Peter Tonner

Jennifer Wygoda

Works CitedSchmid, A. K., David, R. J., Pan, M., Kolde, T., & Baliage, N. S. (2009). A single transcription factor regulates evolutionarily diverse but functionally linked metabolic pathways in response to nutrient availability. Molecular Systems Biology,5(282), 1-15. doi: 10.1038/msb.2009.40.