Abstract

Red sea, described as one of the unique marine ecosystems, incorporates up to 25 deep-sea brine pools. These pools posses multiple extreme conditions influencing the evolution and survival of their inhabiting microbial community. The combination of maximum depth (2194 m), high temperature (68C), anoxia, high salinity (26%), high pressure and high concentrations of heavy metals in the lower convective layer (LCL) of the Atlantis II brine pool makes it an ideal environment for identification of novel enzymes with unique characteristics and potential biotechnological applications. Here we describe the identification and the preliminary in vivo functional investigation of the ligase domain of an ATP-dependent DNA ligase from the DNA of the prokaryotic community extracted from water samples of the LCL of Atlantis II brine pool. Previously, these water samples were serially filtered on different membranes and the DNA isolated from the 0.1­m filter was subjected to 454 pyrosequencing. A metagenomic dataset was initiated and used in this study to mine for genes encoding DNA ligases through Pfam search of conserved domains. The search and subsequent bioinformatic analysis resulted in the identification of a contig harboring an ORF of 915 bp (305 amino acids) that encodes a putative DNA ligase (LigATII). Homology search of the putative DNA ligase showed highest similarity to Erysiopelotrichaceae Bacterium (39% identity, 54% positive). LigATII displays modular architecture that is similar to two distinct domains-(the adenylation domain of LigD and the oligonucleotide binding (OB) fold domain)-that are conserved in ATP-dependent DNA ligases. Functional annotation of the LigATII ORF, identification of the functional conserved amino acids by the Consurf tool, 3D modeling and comprehensive phylogenetic analysis were conducted. These analyses have revealed the relatedness of LigATII to the family of ATP-dependent DNA ligases that has been recently identified through computational studies to exist in prokaryotes. This family is expected to be involved in the specialized form of genomic DNA repair through the non-homologous end joining pathway which acts to join double-stranded breaks (DSBs) or to promote genetic diversity under conditions of selection pressures. Accordingly, the putative LigATII was amplified from the whole genome DNA amplification of LCL. Sanger sequencing confirmed the sequence of the gene before cloning into pET100 Topo directional expression vector. The cloned LigATII was transformed into a temperature sensitive mutant strain of Escherichia coli; strain GR501, with mutation in the DNA ligase gene. LigATII complemented the temperature sensitive strain at the non-permissive temperature (43â—¦C) verifying the in vivo functional activity. The biochemical characteristics of the novel LigATII protein will be described.

Department

Biotechnology Program

Degree Name

MS in Biotechnology

Graduation Date

6-1-2014

Submission Date

May 2014

First Advisor

Siam, Rania

Committee Member 1

Aziz, Ramy

Committee Member 2

Mohammed, Wael

Extent

79 p.

Document Type

Master's Thesis

Library of Congress Subject Heading 1

Biotechnology -- Red Sea -- Egypt.

Library of Congress Subject Heading 2

Molecular biology -- Red Sea -- Egypt.

Rights

The author retains all rights with regard to copyright. The author certifies that written permission from the owner(s) of third-party copyrighted matter included in the thesis, dissertation, paper, or record of study has been obtained. The author further certifies that IRB approval has been obtained for this thesis, or that IRB approval is not necessary for this thesis. Insofar as this thesis, dissertation, paper, or record of study is an educational record as defined in the Family Educational Rights and Privacy Act (FERPA) (20 USC 1232g), the author has granted consent to disclosure of it to anyone who requests a copy.

Institutional Review Board (IRB) Approval

Not necessary for this item

Comments

First the person who influenced me the most in my graduate career has been my advisor Dr. Rania Siam, Associate Professor and Chair of the Biology Department in the School of Science and Engineering at the American University in Cairo. I am thankful to my thesis committee members. Thanks for Dr. Wael Mohammed, Visiting Assistant Professor in the Biology Department at the American university in Cairo, Dr. Ramy Aziz, Assistant Professor in the Microbiology and Immunology Department at the Faculty of Pharmacy Cairo University and Dr. Ahmed Abdellatif, Visiting Assistant Professor in the Biology Department at the AUC, Thanks to all of them for agreeing to serve in my committee. I would like to thank all the professors in the department who taught me through my master program and also I would like to thank the administrators in our school division for providing any assistance requested. I acknowledge the King Abdullah University of Science and Technology (KAUST) for funding the Red Sea Metagenomics project and for the graduate research fellowship that provided the necessary fincial support for this research. I am grateful to hla Hussien her for helping me to learn that there is more than one way to approach a problem. I would also like to give a special thanks to my colleagues and friends in the American University in Cairo. I am especially grateful to Hadeel El-Bardisy and Aya Medhat who I had the great fortune of becoming close friends. I thank my lifetime friends outside the AUC for being truly amazing friends. Last, but by no means least, to thank the people who made me into the person who I am now, my Mom and Dad, I love you both and I wish you all the happiness. My sister and my best friend Maram, thank you for your support through my endless favors. Filly, My dearest husband Mohammed, you are my one and only, I could not have done all of this without you by my side and my precious daughter La, I have done this for you to be proud of your mother. Both of you and your father were my best cheerleaders :) I love you.

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