POSSIBLE CONTRIBUTION OF λ PROPHAGE GENES TO LETHALITY OF Shewanella Oneidensis IN RESPONSE TO CR (VI) STRESS




Saumil Sethna

Introduction

Shewanella oneidensis is a Gram bacteria which can utilize various organic and metal compounds. Compounds utilized include: uranium, chromate, technetium, and nitrates which can be toxic to humans and other organisms The metal oxyanion chromate is a widespread environmental contaminant due to its prevalent use in industrial and defense applications such as tanning, electroplating, paint pigment manufacturing, stainless steel welding, and nuclear weapons production (James. 1996; Langard. 1980). The hexavalent form of chromium, Cr (VI), is highly soluble and toxic, with chronic exposure leading to mutagenesis and carcinogenesis (Mazdak et al. 2009). The other most stable, common form of chromium, trivalent Cr (III), is not a carcinogen (Plaper et al. 2002).

Shewanella can convert chromium (VI) dissolved in contaminated groundwater to Cr (III), which is incapable of dissolving in water, preventing Cr (VI) (Lovley et al. 1991) from spreading as the groundwater flows. This ability makes Shewanella an important factor in confining and cleaning up contaminated areas. It can grow naturally almost anywhere, and does not cause disease in humans or other organisms. These properties make it an ideal bacterium for bioremediation of contaminated environments.

S. oneidensis genome has a λ phage integrated into it (prophage). λ phage has two potential life cycles; lytic and lysogenic. If it goes into the lytic cycle it lyses the bacterial cell. On the other hand if it proceeds to the lysogenic cycle, it integrates into the bacterial chromosome and replicates along with it, without causing harm to the bacteria (Dodd et al. 2005). Prophage gets induced in response to stresses such as Cr (VI) exposure. On induction of the lytic pathway, it forms the phage particles and lyses the bacteria (Friedman. 2005).

The primary goal of this project was to gain insight into the changes in the mRNA expression patterns of λ prophage structural genes in response to either acute (90 min.) or chronic (24 hrs.) Cr (VI) (Chourey et al. 2006) stress in S. oneidensis cells. Prophage LambdaSo major tail protein V (PTP) and Prophage LambdaSo major capsid protein (PCP) were chosen as genes of interest.

Figures


Figure 1-Genes Studied and Primer Information


Figure 2-Gel pictures of RT-PCR products for PTP. Lanes 1, 3 and 5 are untreated (U) and lanes 2, 4 and 6 are treated (T) with Cr (VI) for different times. Lane 7 is negative control. Gap- A1 is the loading control.


Figure 3-Gel pictures of RT-PCR products for PCP. Lanes 1, 3 and 5 are untreated (U) and lanes 2, 4 and 6 are treated (T) with Cr (VI) for different times. Lane 7 is negative control. Gap- A1 is the loading control.


Figure 4-Products detected by RT-PCR are due to RNA and not DNA contamination


There is a basal expression level of PTP transcript but not PCP transcript. Cr (VI) treatment, neither acute (1mM, 90 min) nor chronic (0.3mM, 24 hrs.), seems to have an affect on the expression level of PTP. On the other hand PCP seems to be induced after chronic but not acute Cr (VI) treatment. The bacterial culture enters into the stationary phase by ~24 hrs. (Brown et al. 2006) There is no detectable difference in the expression levels of PTP in response to different growth phases of the bacteria. PCP is induced only due to Cr (VI) stress and not in response to the bacteria proceeding to the stationary phase.

PTP alone is not enough to cause the lysogen to go into the lytic cycle but on induction of the capsid protein, it could potentially combine with the tail protein and the other phage proteins to induce lysis of the bacterial cell.

Mutant S. oneidensis without the prophage genes might be able to withstand Cr (VI) stress for a longer duration.

Please refer to the attached paper for full details.

Full Paper

Acknowledgments

I would like to thank Dr. Rubin for obtaining the culture and his continued insights into the project. Special thanks to Bo Liu and Leleesha Samaraweera, who helped and guided me throughout the project and cheered me up when I was struggling.


This document was last modified 05/11/2009.
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