Production and Characterization of Extremolytes from Indigenous Radio-resistant Microorganisms and their Evaluation for Potential Biotechnological Applications

By

Wasim Sajjad

Department of Microbiology Quaid-i-Azam University Islamabad, Pakistan 2017

Production and Characterization of Extremolytes from

Indigenous Radio-resistant Microorganisms and their

Evaluation for Potential Biotechnological Applications

A thesis

Submitted in the Partial Fulfillment of the

Requirements for the Degree of

DOCTOR OF PHILOSOPHY IN

MICROBIOLOGY

By

Wasim Sajjad

Department of Microbiology Quaid-i-Azam University Islamabad, Pakistan 2017

DECLARATION

The material contained in this thesis is my original work and I have not presented any part of this thesis/work elsewhere for any other degree.

Wasim Sajjad

DEDICATED

TO

My Ammi, Abbu &

Late Grand Father

CERTIFICATE

This thesis, submitted by Mr. Wasim Sajjad is accepted in its present form by the Department of Microbiology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad as satisfying the thesis requirement for the degree of Doctor of Philosophy (PhD) in Microbiology.

Examiner: ______

Examiner: ______

Supervisor: ______

Dr. Aamer Ali Shah

Chairperson: ______Dr. Rani Faryal

Dated:

CONTENTS S. No Chapter no Title Page no

1 List of Abbreviations i

2 List of Tables iii

3 List of Figures iv

4 Acknowledgements vi

5 Abstract viii

6 Chapter 1: Introduction 1

7 Chapter 2: Review of Literature 14

8 Isolation and Characterization of Ultra Violet 78 Chapter 3: Rays (UVR) Resistant from Desert Soil Samples of Pakistan 9 Chapter 4: In-Vitro Cytotoxic and Antioxidant Activities of 112 Extremolytes from Radio-Resistant Bacteria

10 Chapter 5: Radio-protective and antioxidative activities of 132 astaxanthin from newly isolated radio-resistant bacterium sp. strain WMA-LM9

11 Chapter 6: Ectoine: a Compatible Solute in 165 Radiohalophilic Stenotrophomonas sp. WMA LM19 Strain to Prevent -Induced Protein Damage 12 Future prospectives 196

13 I Appendices 197

II Turnitin Origiility Report 216

III Published Research Article 219

List of Abbreviations A Adenosine ATP Adenosine-5’-triphosphate AUG 30 Augmentin 30µg BSA Bovine serum albumin BLAST Basic Local Alignment Search Tool BLASTN BLAST search using a nucleotide query bp Base pairs C Cytosine °C Degree celsius CAT Catalase CTX 5 Cefotaxime 5µg DNA Deoxyribonucleic acid dNTP Deoxynucleoside triphosphate DPPH 2,2-Diphenyl -1-picrylhydrazyl DTNB 5,5’-Dithio-bis 2-nitrobenzoic acid DEPPD N,N- Diethylparaphenyldiamine DNPH 2, 4- Dinitrophenyl hydrazine DNS Dinitrosalicylic acid EDTA Ethylenediaminetetraacetic acid et al. et alii/alia, and others e.g. Exempli gratia, for example Fe Iron Fig. Figure FTIR Fourier transform infrared spectroscopy GR Glutathione reductase

G Guanine HPLC High-performance liquid chromatography i.e. id est, that is IMI 10 Imipenem 10µg LCMS Liquid chromatography mass spectroscopy MDA Malondialdehyde MDR Multidrug resistant NBT Nitrobluetetrazolium NMR Nuclear magnetic resosnance NaCl Sodium chloride NCBI National Center for Biotechnology Information

OD Optical density/ Absorbance PCR Polymerase chain reaction pH Power of Hydrogen POD Peroxidase rRNA Ribosomal ribonucleic acid

Rf Retardation factor ROS rpm Rotation per minute SDS Sodium dodecyl sulfate SDS PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SOD Superoxide dismutase SXT 25 Trimethoprim-sulphamethoxazole 25µg T Thiamine TBA Thiobarbituric acid TCA Trichloroacetic acid TGC 15 Tigecycline 15µg TGY Tryptone yeast extract TLC Thin layer chromatography TPP Tripolyphosphate U Uracil

List of Tables No. Title Page No. 2.1 Ultraviolet radiation (UVR) resistance in variety of radio-resistant micro- 21 organisms. 2.2 Ultraviolet radiation (UVR)-inductive microbial metabolic products and 22 their therapeutic implications. 3.1 Shows culture code, Sampling site, Colony morphology and Gram 92 staining of UVR isolates from desert soil. 3.2 Biochemical and physiological characteristics of UVR isolates from Lakki 94 Marwat and Bahawalpur desert soil. 3.3 16S rRNA sequence homologues, closest related species, % 95 survivability, gene bank accession number and query coverage of Ultraviolet radiation (UV subtype –B) resistant isolates from desert samples. 3.4 Effect of the metal ions (in ppm) on growth of UVR resistant selected 98 bacteria from desert samples on TGY agar plates. 4.1 Anti-microbial activity of partially purified extracts from UV resistant 124 microbes.

List of Figures

No. Title Page No. 2.1 Diagrammatic representation of origin of different types of radiation and 20 their effects on 2.2 Carotenoid biosynthetic pathway showing biosynthetic enzymes and 28 intermediate product with Deinoxanthin as the final product. 2.3 Different pathways for DNA double-strand break repair 34 2.4 Two stages of genome reconstitution in 35 2.5 Main pathways involved in the processing of DNA ends. 39 2.6 Screening strategy for bio-active compounds from extremophiles. 43 2.7 Structure of Scytonemin, a novel dimeric molecule (molec. wt. 544) of 46 indolic and phenolic subunits 2.8 Production and hypothetical pathway in which scytonemin inhibits 50 PLK1 3.1 Metal analysis (in ppm) of soil samples collected from deserts 93

3.2 Survivability of total UVR resistant isolates from desert soil at varying UV- 93 B exposure. 3.3 Phylogenetic analysis UV resistant bacterial strains by maximum likelihood 96 method 3.4 Effect of UVB on total cell protein content in mg/ml. 97 3.5 Protein oxidation to quantify carbonylated protein and lipid peroxidation 99 assay for TBARS in UV treated isolates from desert soil.

4.1 The fluorescent quenching spectrum of methanolic extracts 1mg/ml from 120 UVR bacteria 4.2 The cytotoxic effect of partially purified fractionated extracts on HeLa cell 122 line by using MTT assay. 4.3 Comparative analysis of IC50 values of different fractionated extracts of 123 UV resistant microbes. 4.4 Anti-microbial assay of partially purified extracts from radio resistant 123 bacteria. 5.1 Astaxanthin synthesis from isophorone, cis-3-methyl-2-penten-4-yn1-ol 135

and a symmetrical C10-dialdehyde has been discovered and is used commercially. 5.2 Structure of deinoxanthin from Dienococcus radiodurans. 136 5.3 Structure of phenolcs and flavonoids 136 5.4 Survivability of strain WMA-LM9 from desert soil at varying UV-B 145 exposure. 5.5 Comparison of intracellular protein carbonylation level between radio- 148 resistant Deinococcus sp. strain WMA-LM9 and E.coli (10536)

5.6A Flash chromatography of the extract using different solvent system of 149 Hexane Dichloromethane water and methanol. 5.6B Different fractions collected upon flash chromatography for bioassay 149 guided fractionation. 5.7 HPLC chromatogram/positive ESI-MS spectrum of carotenoid extract 150

5.8 1H NMR spectra of purified compound LM9F1 150 5.9 13C NMR spectra of purified compound LM9F1. 151 5.10 Astaxanthin chemical structure from NMR peaks using ChemDraw 151 software.

5.11 Anti-oxidant activity of carotenoid extracted from strain WMALM9. 153

5.12 Inhibitory effect of carotenoid from strain WMA-LM9 of different protein 154 oxidation in-vitro. 5.13 Role of carotenoids in prevention of oxidative damage to pUC18 plasmid 155 DNA after exposure to oxidative agents. 6.1 Structure of some compatible solutes from extremophiles 167 6.2 Survivability of strain WMA-LM19 from desert soil at varying UVB 176 exposure. 6.3 Neighbor joining phylogenetic tree based on 16S rRNA gene sequence 177 analysis, showing the position of isolate WMA-LM19 6.4 HPLC chromatogram/positive of the polar extract for WMA LM19 177 6.5 ESI-MS spectrum of purified ectoine extract 179 6.6 1H NMR spectra of purified compound LM19F2. 179 6.7 13C NMR spectra of purified compound LM19F2. 180 6.8 Chemical structure of ectoine from NMR spectroscopy. 180 6.9 DPPH and OH Free radical scavenging assay using ascorbic acid as positive 183 control 6.10 Assay of reducing power and Fe chelation of ectoine using ascorbic acid 183 and EDTA as positive control 6.11 Protein oxidation and lipid peroxidation inhibition activity offered by 184 ectoine 6.12 Membrane damage preventing assay by ectoine using human RBCs and 185 Lecithin as positive control 6.13 Analysis of protein protection offered by ectoine on SDS-PAGE. 186

Acknowledgements

Praise to ALMIGHTY ALLAH, whose blessings enabled me to achieve my goals. Tremendous praise for the Holy Prophet Hazrat Muhammad (Peace Be upon Him), who is forever a torch of guidance for the knowledge seekers and humanity as a whole.

In front of you lies the result of my PhD research at the Quaid-i-Azam University Islamabad, Pakistan. A period in which I have learned so many things: not only about radio-resistance and bacteria, but even more about ourselves and each other. This research proposal represents that period, our first –humble- steps on the stage of science. It was a time full of

surprises: the uneasy but cosy introduction, the path from our starting point to the radio- resistant microbes, and above all, the taste of a genuine scientific research project.

All of this would not have been possible without the help of numerous smart people. I would therefore like to thank the following people sincerely for the time and effort they put into our project: Prof. Dr. Fariha Hasan, Dr. Javid Iqbal Dasti, Dr. Samiullah Khan, and. Dr. Arshad Jhangir for the extremely useful suggestions and guidelines during our research work.

I do not find enough words to express my heartfelt gratitude for Dr. Kerry McPhail, Associate Professor, Department of Pharmaceutical Sciences Oregon State University, USA. She supervised me during my studies in Oregon State University during International Research Support Initiative Program (IRSIP). This experience would not have been as valuable without the guidance, support and inspiration provided by her. I am impressed by her scientific thinking and politeness. I am also thankful to Celick, David, Richard and Nazir Muhammad, Postdoctoral Research Associates at Department of Pharmaceutical Sciences, for their care and immense help during my entire stay at Oregon State University.

I would also like to thank Higher Education Commission, Pakistan, for providing me grant under the Project “IRSIP” to work in multicultural dynamic environment with internationally recognized scientists.

A non-payable debt to my loving Ammi (Mushtari BiBi), Abbu (Muhammad Pervaiz), brothers (Shahid Iqbal, Fawad Alam, Muhammad Abbas and Waqas Ahmad) and sisters for bearing all the ups and downs of my research, motivating me for higher studies, sharing my burden and making sure that I sailed through smoothly. Completion of this work would not have been possible without the unconditional support and encouragement of my loving family members. I would like to acknowledge my Uncle Iqbal Ahmad Tajik and cousins Irshad Alam. Mukhtiar Alam (My school mentor), M. Idrees, Shahab Ahmad for their support.

I extend my greatest depth of loving thanks to all my friends and laboratory mates (seniors and juniors) especially Muhammad Rafiq, Muhammad Irfan, Imran Khan, Sahib Zada, Abdul Haleem, Arshad, Abdul Haq, Ghufran, Matiullah, Waqas, Wasim Hukam, Manzoor, Umair, Akhtar Nadhman, Faiz, Saad, Shahid, Barkat, Faiz, Shama, Maliha, Nazia Anum and Mahvish, Adnan Khan, Asim Shah, Adil Nawaz, Izhar for their help throughout my study.

Special thanks to a cluster of people, always with me in my ups and down during PhD. These special people are Manzoor Ahmad, Salman Khan, Sunniya Ilyas, Sundas Qadir, Maryam Zeest Hanif and Misbah, MSc students Anum, Saiqa and Shaista. I especially want to express

a heartfelt gratitude to Fariha Aman Nazir, whose sincere encouragement and understanding attitude are assets worth cherishing forever.

There is, however, one person who stands out among all these people: Dr. Aamer Ali Shah, my mentor. I would like to thank him for the time he spent at our meetings and assessment evenings, for the input he gave me during the process and especially for the fact that he, despite my project being way out of his research area, has always helped me throughout the entire period.

Finally, I express my gratitude and apology to all those who provided me the opportunity to achieve my endeavors but I missed to mention them personally.

Wasim Sajjad

Summary

Radio-resistant microorganisms have the puzzling ability to withstand with high range of extreme conditions including high doses of ultraviolet radiation and desiccation. Radiation in general has the ability to cause extensive damage to various cell components such as nucleic acids, proteins and membranes. Nevertheless, metabolic products such as extremolytes facilitate the survival of microorganisms in such extreme conditions. These extremolytes are not essential for the organism in terms of its normal growth. However, it provides a competitive edge to the organism in terms of survivability against stress and assist in regulating reproductive responses. Thus far, the use of radio-resistant extremophiles as a source of natural products has largely been ignored in modern biotechnology. The current study focused on the isolation of radioresistant bacteria from extreme environment. The phylogenetic diversity of ultra-violet (UV) resistant bacteria from deserts soil, was investigated by culture and molecular based analysis. The bacterial strains were observed for their tolerance to UV doses, salt concentration, and heavy metals. The effect of UV radiation on cellular protein and lipids was also investigated. The secondary intracellular bio-active compounds extracted from these radio resistant bacteria, were assayed for cytotoxic, antioxidant and antibacterial activities in order to evaluate their potential to be considered as therapeutic and radio protective agents. Based on higher UV resistance and production of higher amount of UV absorbing compounds, two strains designated as WMA-LM9 and WMA- LM19 were selected for further studies. Two different compounds were extracted in a solvent system and purified by highperformance liquid chromatography (HPLC) on a C18 analytical column. The compounds were characterized as mono-esterified astaxanthin and ectoine by 1H, 13C nuclear magnetic resonance (NMR) and mass spectrometry (MS). These pure compounds were tested for antioxidant activity, total flavonoids and phenolic content, radio protective potential in correlation to the prevention of protein oxidation and DNA strand breaks in-vitro. The antioxidant and radio-protective properties of the pure

compounds were evaluated by hydroxyl scavenging, 2,2-diphenyl-1picrylhydrazyl (DPPH) reducing, lipid peroxidation inhibition assays and protein radio-protection assays by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis.

A total of 09 UV resistant bacteria were isolated and identified through biochemical tests and 16S rRNA gene sequencing. Based on the results obtained, bacterial strains were assigned four phyla: Firmicutes, Proteobacteria, DeinococcusThermus and Actinobacteria. High UV survivability was observed in case of genus Deinococcus followed by Firmicutes. The bacteria were found to grow at wide temperature and pH range, resistant to high salt concentration as well as various metal ions. The bacterial strains exhibited minor damages to protein and lipids as a result of exposure to UV radiation as compared to Escherichia coli (ATCC 10536). The purified carotenoid pigment, Astaxanthin from Deinococcus sp. strain WMA-LM9, also showed a higher inhibitory action against oxidative damage to collagen, elastin and bovine serum albumin than a standard compound, β-carotene. It also inhibited breaks to DNA strands, as indicated by the results of the DNA damage prevention assay. Another compatible solute, Ectoine, purified from strain WMA-LM19, exhibited strong Fe2+ chelation in comparison to EDTA (38.58± 0.84%). The OH- radical scavenging efficiency of ectoine (53.68 ± 0.48%) was estimated in terms of % inhibition of deoxy D-ribose degradation in a non-site-specific assay using a concentration of 10.0 μg/ml. Maximum reduction in DPPH (~60.45 ± 1.185%) was observed at 10 μg/ml ectoine concentration. Ectoine effectively inhibited oxidative damage to proteins and lipids in comparison to the standard ascorbic acid. Furthermore, a high level of ectoine-mediated protection of bovine serum albumin against ionizing radiation (1500-2000 Jm-2) was observed, as indicated by SDS-PAGE analysis.

The results indicated that these radio-resistant microbes harbor a sophisticated phenotypic character and molecular repair mechanisms that can prolong their survival in extreme radiations. The current research work concluded that the radio-resistant strains from extreme environment have great potential to produce potent metabolites with a wide range of antioxidant and cytotoxic activities. The extremolytes showed a good radio-protective effects against radiation-mediated cell damage, and were considered as a potential mitigator to overcome oxidative damages in extreme environment. These extremolytes can potentially be harnessed as radio-protective drugs and can also be used as sunscreen to block UV radiations.

1. Introduction

This environment manifests itself as any of two versions; moderate or extreme. Environments with a limited number of nutrients and water supply, such as deserts, or others with highly saline waters such as lakes with high salinity constitute examples of such extreme environments. The term was put to paper roughly a quarter century ago (MacElroy, 1974). Extremophiles are the microbes that can survive in harsh or extreme condition of pressure, salinity, temperature or in concentrations of other chemicals that would kill other microbes or creature. Although a varied number of definitions for the term exist, they all refer to organisms that can thrive in such environments that are otherwise uninhabitable to other organisms. Twenty years ago extremophiles were considered cheap and useless organisms and were explored by very few scientists, but nowadays they are used as a source of novel compounds and extremozymes (Schulze-Makuch et al.,2015).

Radio-resistant microbes constitute a group of extremophiles that have the ability to survive environments with a high level of radiation (Singh et al.,2013). Radio-resistant microbes too, in accordance with the definition of extremophiles, thrive under conditions which are otherwise hostile to other organisms; in this case, the extreme conditions are high levels of radiation. The phenomenon of tolerance to radiation is known as radio-resistance. All three domains of life, namely Bacteria, and can exist as radio- resistant extremophiles. These radioresistant bacteria carry reactive oxygen species (ROS)- scavenging enzymes and exhibit diverse metabolic properties (Yu et al.,2015). The search for radio-resistant species and mechanisms resulting in higher, radio-resistance in certain several species, all of which are competent to withstand huge amounts of radiation. Although the earliest list included organisms from three distinct domains (Farmilo et al.,1973), including the following four species.

Chroococcidiopsis sp.: This is the most primitive cyanobacteria. It can survive in harsh environmental conditions, both in high and low temperature. It can also survive in high salinity and ionizing radiation. They have the potential to withstand gamma radiation as high as 15 kGy without undergoing mutation (Billi et al., 2000).

Deinococcus radiodurans: One of the best known radio-resistant specie. This bacterium has the unique ability to repair damaged DNA and is recorded as the maximum resistant life form (as of May 2012) in the Guiness Book of Records. They possess two primary factors to tolerate lethal radiations i.e. carotenoids and efficient DNA repair pathways.

Rubrobacter radiotolerans: This is a relatively unknown bacterium, resist UV rays by two different protecting mechanisms. This bacterium partially known to have superoxide dismutase and carotenoids producing ability to survive in extreme environments (Ferreira et al.,1999; Terato et al.,2011).

Thermococcus gammatolerans: This archaea clustered in order of Thermococcales and without apparent lethality, specie can withstand a dose of 3 kGy, but the exposure to a higher dose of radiations can only slightly reducing its viability (Tapias et al.,2009).

Radiations are the energy in the form of electromagnetic waves. Elements with unstable isotopes decay spontaneously and can ultimately form other toxic elements. They radiate radiations (ionizing or may non-ionizing) during this process. These radiations have a great potential to eject electrons from the outermost orbital of the atoms. All this results in the formation different ions (with having great ionization power) which can charge the other molecules (Kumar et al., 2010). UV radiation (UVR), can form different dimers in DNA by changing its molecular bonding and structure between DNA strands. Radiation in general has the ability to cause extensive, complex and lethal damage to various cell components such as nucleic acids, proteins and membranes. Free radicals produced by such radiation as it passes through the cell, directly or indirectly, are responsible for the damage caused to the cellular components (Santos et al., 2013). These can undergo different metabolic damages due to superoxide’s generation if exposed to the right amount of radiation, such as ultraviolet (UV) rays, Gamma Rays or X-rays, owing to the induction and the consequent presence of a variety of cytotoxic and mutagenic DNA lesions. Examples of damage at the

DNA level, brought on by radiation be composed of single and double-strand breaks, impaired purine and pyrimidine bases, subtraction of bases and cross-linkage formation between adjacent protein molecules and DNA (Upadhyay et al., 2005). The most extreme effect of irradiation is protein oxidation and lipid peroxidation in biological membranes, especially mitochondrial membranes (Tian et al., 2009).

The two different mechanisms that render organisms radio-resistance: protection and repair. In the first case the antioxidant defense system protects the cell vital constituents from direct or indirect damage by ionizing radiation. In the second case DNA damage is followed by well developed a repair mechanism which mends DNA lesions quickly and efficiently. Examples of this mechanism exist in several distinct organisms. Earth molecules receives a numerous amount of radiation continuously marked from the Earth’s crust and space. These organisms need to refine a huge radio-resistant, but on the hot and dry deserts the dose is significantly above the average. The survival of radio-resistant microbes to tolerate radiation is based on the development of sophisticated radio-resistant mechanisms. In biology and medicine, the consequences of proteome damage on the maintenance of life are highly underestimated (Krisko and Radman, 2013). Premature aging and cancer are the consequences of protein and lipid modifications brought on by stress or other environmental factors. UVR resistant microbes enjoy a largely effective protection against proteome damage. However, this protection does not extend to their genome. The well protected proteome not only counters the effects of extreme radiation damage, by facilitating molecular repair but its production for extremolytes play an central role in the survival.

In light of the above findings, various therapeutic agents have been studied that may reverse the effects of radiation injury in the target organism. The effects of these agents are as follows:

1. Reduction in free radical induced 2. Repair of oxidative damage and recovery enhancing 3. Modifications in the immune system (biological-response modifiers) (Ghose 1983; Nair et al., 2004)

Nevertheless, metabolic natural products such as extremozymes and extremolytes facilitate the survival of microorganisms (Gabani and Singh, 2013). Thus far, the use of radio- resistant extremophiles as a source of natural products that may be used as drugs has not been studied enough. However, from the limited research that has been conducted on products obtained from radio-resistant microbes, we now know that in addition to being used as tools across a range of potencies, radio-resistant possess a range of beneficial properties such as: lowering cholesterol, immunosuprresion, antiparasitic abilities, herbicidal, used as diagnostic tools, antibacterial, antifungal properties and antiviral activities.

Extremolytes are secondary organic molecules with low molecular weight possessing a range of frequently very potent biological activities. These metabolites are not essential for

the organism in terms of its normal growth, development or reproduction. They do, however, give a competitive edge to the organism to help in its survival against other inter- species competitors, provide protection against stress and assist in regulating reproductive responses (Mandal and Rath, 2015). Success has been achieved in the search for radio- resistant extremophiles that yield extremolytes which have potential as therapeutic and protective agents. So far, the isolation of several UVR-protective compounds obtained from UVR-resistant extremophiles has been successful, including: ectoine a UV protectant (Beblo- Vranesevic et al., 2017), scytonemine, mycosporine-like amino acids (MAAs), bacterioruberin, pannarin, melanin and many others (Ferroni et al., 2010). Some of these compounds have shown to be instrumental in the treatment or prevention of a myriad of diseases, many of which had no known cure prior to the discovery of these compounds.

These extremolytes obtained from various extremophiles including cyanobacteria, act as antioxidants and have offered protection against oxidative stress in human cells, proteins and lipids (Rastogi et al., 2009; Singh et al., 2013; WaditeeSirisattha et al., 2016). Manganese-metabolite mixtures, yielded from the environmentally resilient D. radiodurans species, display excellent antioxidant properties that have been studied with effective role in offering protection to different cell lines against oxidative damage. These organisms, thus appear promising in delaying the process of aging, working against other age-related diseases as well as preventing cancer. The challenge that remains in the near future is to utilize these extremophilic antioxidants or extremolytes against aging and cancer-related DNA as well as protein modifications (Slade and Radman, 2011).

The attention of several industrial sectors, especially the cosmetics and pharmaceutical industries, has turned to these extremolytes because of the association that exists between their biological activities and the various health benefits offered (chronic disease prevention, activity against carcinogenic agents etc.). Although their industrial significance has been significantly exposed, much light remains to be shown on their significance as therapeutics. The unique metabolism of these microbes, in addition to being responsible for great diversity amongst the microbes, also facilitates the ability of these microbes to survive in harsh environments (Ferrer et al., 2007; Gostincar et al., 2010). The demand for carotenoids in the food, feed and pharmaceutical industries is ever-growing and can be met by employing microorganisms using biotechnology (Galano et al., 2010).

Radio-resistant extremophiles hold great importance as biological materials and as an enzyme source in regards to their biotechnological applications globally, in addition to their role in structural and biochemical biodiversity (Jane and Alan, 2004; Podar and Reysenbach, 2006). The discovery of radio-resistant extremophiles and their associated bio products and their industrial use show great promise in advancing mankind’s advances. The development of innovative or better drugs has been facilitated by an increased understanding of the roles that these microorganisms play in these processes, coupled with our ability to manipulate their activities using molecular biology techniques.

Genome stability is known to be one of the important factors for UV resistant bacteria to survive in extreme environments, but in response to sever UV radiations they are also capable of producing different primary and secondary metabolites (Makarova et al.,

2001; Bagwell et al., 2008; Goosen and Moolenaar, 2008; Singh and Gabani, 2011). The secondary metabolites are produced for their own defense in these extreme environments and are yet to be investigated for mechanistic intervention (Carreto and Carignan, 2011). The recent biotechnological applications may aid to pinpoint the adapted microbial approach of self-engineering to oppose under extreme UVR. Therefore, it is necessary to determine the diversity of UVR- resistant microorganism from the natural environment on earth to explore the physiological mechanism adapted by microorganism to withstand extreme UVR. It could be justified that the hot and dry environment with limited water availability and nutrients would divulge a diversity of UVR resistant extremophiles with regulated proteins/enzymes. Therefore, we pursue to segregate and characterize the diverse variety of microorganisms resistant to UVR isolated different desert soil of Pakistan. The protection based radio-resistant bacteria that were found in a dry and hot desert of Pakistan was mainly focused in this report. By comparison with Deinococcus radiodurans that is considered as model specie in radio-resistance studies, the question raised was whether all of these UVR resistant bacteria contain any carotenoids and compatible solutes like structure that are strong anti-oxidant found in extremophiles. Modern biotechnological techniques could play a vital role in induction or activation of biosynthesis of bioactive compounds cellular metabolites or radio responsive pigments to provide an opportunity for the other organism to survive under radiation rich environment. Genes that produce the metabolites that can be protective against radiation could be induced by growing UV resistant organism in the presence of UV-light (Singh and Gabbani, 2011).

The hot and dry environment with high UV radiation has largely been ignored by scientists in Pakistan. To the best of our knowledge, there is no report on production of extremolytes from radio-resistant microbes in Pakistan. The research work presented in this thesis includes 6 chapters (including this introduction and literature review as chapter 1 and 2). The second chapter describes the diversity, and adaptation mechanisms of radio- resistant bacteria in high radiation as mentioned in separate sections of literature review (see chapter 2). The current study focuses: (a) Isolation and characterization of UV resistant bacteria from the extreme environment of Pakistan. Effect of UVB on cell proteins and lipids was also investigated in comparison to E. coli, (UV sensitive ATCC strain 10536) that has been subjected to UV radiations (see chapter 3). (b) Extraction of extremolytes and its partial purification which is then subjected to various bio-assays such as measurement of cytotoxic and antioxidant activities. The main focus here was to screen out the potent UV absorbing and radio-protective compounds (see chapter 4). The main subjects were Deinococcus sp. strain WMA-LM9 and Stenotrophomonas sp. strain WMALM19, isolated from desert soil samples of Pakistan and characterized via 16S rRNA sequencing (c) The fifth chapter (paper 3) deals with the extraction and purification of astaxanthin from Deinococcus sp strain WMA-LM9. A purified astaxanthin was quantified and assayed for different anti-oxidative and radio-protective activities. (d) We further selected another radio-halophillic strain Stenotrophmonas sp. WMALM19. A compatible solute, ectoine was extracted from this radio-halophillic bacterium Stenotrophomonas sp. WMA-LM19. It was purified and its potential role as radio-protective and anti-oxidative agent in vitro has been discussed in chapter 6 (paper 4). Deinococcus and Stenotrophomonas species possess very strong molecular repair mechanisms which have been investigated previously by many scientists.

The protection mechanism against free radical provides resistance to microbe by producing antioxidant carotenoids and compatible solutes ectoine that offers protection against UVB.

This study will come up with implications such as the discovery of a unique environment for UVR microbes in the context of Pakistan, source of novel extremozymes and extremolytes as a source of therapeutic agents with potential industrial applications. These results will also open the exiting standpoints on investigating bacterial lenience to desiccation, radiation and survey in the deserts. This report tries to put one of these mechanisms in the spotlight that is the production of different compounds with radio- protective and anti-oxidative activities. These compounds can act as a primary defense mechanism and play positive part in the survival of radio-resistant microbes in extreme UV radiations and thus can prolong their survivability. The experimental design and the outcome of this research proposal will contribute information on the radio-resistance of the specified microorganisms.

1.1. Research Hypothesis

The UV resistant organisms produce carotenoids and UV absorbing compounds that protect the bio-molecules from excessive damage due to UV exposure and extend their survival. These compounds however act as superoxide quencher that can detoxify the unstable toxic molecules which leads to tissue protein and lipid damage. The extremolytes or the bioactive compounds produced by radioresistant microbes can prolong the survival of these extremophilc bacteria in a high UV exposure. These compounds can act as melanin in human that prevent the entry of UV radiation to body and thus acting as a primary line of defense in UV exposure to prevent the cells from excessive damages. Based on our research hypothesis, we want to investigate anti-oxidative strength of extremolytes and carotenoids produced during cell stress.

1.2. Research Questions 2. Do UVR microbes possess carotenoids or other bio-active compounds? 3. How they help in cell survival under extreme stress conditions like high UV dose, desiccation and high salt concentration? 4. Could we consider these bio-active compounds as potential candidates for biomedical applications?

1.3. Aim and Objectives of the Study Aim

The aim of this study was to isolate radio-resistant microorganisms from soil as well as to investigate the role of extremolytes in their survival to UV radiation.

Objectives

The aim has been accomplished through the following objectives.

1. Isolation and characterization of radio-resistant from the desert soil. 2. Extraction and characterization of the UV absorbing compounds that might be involved in their resistance to UV radiation. 3. Chemical and structural analysis of these metabolites using LC-MS/MS, proton and carbon NMR. 4. Bio-assays of these metabolites in order to confirm their role in cellular protection under UV radiation stress.

References 1. Bagwell, C.E., Bhat, S., Hawkins, G.M., Smith, B.W., Biswas, T., Hoover, T.R., Saunders, E., Han, C.S., Tsodikov, O.V. and Shimkets, L.J., 2008. Survival in nuclear waste, extreme resistance, and potential applications gleaned from the genome sequence of Kineococcus radiotolerans SRS30216. PloS one, 3(12), p.e3878. 2. Beblo-Vranesevic, K., Galinski, E.A., Rachel, R., Huber, H. and Rettberg, P., 2017. Influence of osmotic stress on desiccation and irradiation tolerance of (hyper)- thermophilic microorganisms. Archives of Microbiology, 199 (1), pp. 17-28. 3. Billi, D., Friedmann, E.I., Hofer, K.G., Caiola, M.G. and OcampoFriedmann, R., 2000. Ionizing-radiation resistance in the desiccation-tolerant cyanobacterium Chroococcidiopsis. Applied and Environmental Microbiology, 66(4), pp.1489-1492. 4. Carreto, J.I. and Carignan, M.O., 2011. Mycosporine-like amino acids: relevant secondary metabolites. Chemical and ecological aspects. Marine drugs, 9(3), pp.387- 446. 5. Farmilo, A. and Wilkinson, F., 1973. On the mechanism of quenching of singlet oxygen in solution. Photochemistry and photobiology, 18(6), pp.447450. 6. Ferreira, A.C., Nobre, M.F., Moore, E., Rainey, F.A., Battista, J.R. and da Costa, M.S., 1999. Characterization and radiation resistance of new isolates of Rubrobacter radiotolerans and Rubrobacter xylanophilus. Extremophiles, 3(4), pp.235-238. 7. Ferrer, M., Golyshina, O., Beloqui, A. and Golyshin, P.N., 2007. Mining enzymes from extreme environments. Current opinion in microbiology, 10(3), pp.207-214. 8. Ferroni, L., Klisch, M., Pancaldi, S. and Häder, D.P., 2010. Complementary UV- absorption of mycosporine-like amino acids and scytonemin is responsible for the UV- insensitivity of photosynthesis in Nostoc flagelliforme. Marine drugs, 8(1), pp.106-121. 9. Gabani, P. and Singh, O.V., 2013. Radiation-resistant extremophiles and their potential in biotechnology and therapeutics. Applied microbiology and biotechnology, 97(3), pp.993-1004. 10. Galano, A., Vargas, R. and Martínez, A., 2010. Carotenoids can act as antioxidants by oxidizing the superoxide radical anion. Physical Chemistry Chemical Physics, 12(1), pp.193-200.

Page 78

11. Ghose, A., 1983. Protection with combinations of hydroxyl tryptophan and some thiol compounds against whole-body gamma irradiation. International Journal of Radiation Biology and Related Studies in Physics, Chemistry and Medicine, 44(2), pp.175-181. 12. Goosen, N. and Moolenaar, G.F., 2008. Repair of UV damage in bacteria. DNA repair, 7(3), pp.353-379.

13. Gostinčar, C., Grube, M., De Hoog, S., Zalar, P. and Gunde-Cimerman, N., 2010. Extremotolerance in fungi: evolution on the edge. FEMS microbiology ecology, 71(1), pp.2-11. 14. Irwin, J.A. and Baird, A.W., 2004. Extremophiles and their application to veterinary medicine. Irish veterinary journal, 57(6), p.348. 15. Krisko, A. and Radman, M., 2013. Biology of extreme radiation resistance: the way of Deinococcus radiodurans. Cold Spring Harbor perspectives in biology, 5(7), p.a012765. 16. Kumar, M., 2014. Harvesting of valuable eno-and exo-metabolites from cyanobacteria: A potential source. Asian J. Pharm. Clin. Res, 7(1), pp.24-28. 17. Kumar, R., Patel, D.D., Bansal, D.D., Mishra, S., Mohammed, A., Arora, R., Sharma, A., Sharma, R.K. and Tripathi, R.P., 2010. Extremophiles: sustainable resource of natural compounds-extremolytes. In Sustainable Biotechnology (pp. 279- 294). Springer Netherlands. 18. Lademann, J., Meinke, M.C., Sterry, W. and Darvin, M.E., 2011. Carotenoids in human skin. Experimental dermatology, 20(5), pp.377-382. 19. Macelroy, R.D., 1974. Some comments on the evolution of extremophiles. Biosystems, 6(1), pp.74-75. 20. Makarova, K.S., Aravind, L., Wolf, Y.I., Tatusov, R.L., Minton, K.W., Koonin, E.V. and Daly, M.J., 2001. Genome of the extremely radiationresistant bacterium Deinococcus radiodurans viewed from the perspective of comparative genomics. Microbiology and Molecular Biology Reviews, 65(1), pp.44-79. 21. Mandal, S. and Rath, J., 2015. Secondary metabolites of cyanobacteria and drug development. In Extremophilic Cyanobacteria For Novel Drug Development (pp. 23- 43). Springer International Publishing. 22. Nair, C.K.K., Salvi, V., Kagiya, T.V. and Rajagopalan, R., 2004. Relevance of radioprotectors in radiotherapy: studies with tocopherol monoglucoside. Journal of environmental pathology, toxicology and oncology, 23(2).

Page 79

23. Podar, M. and Reysenbach, A.L., 2006. New opportunities revealed by biotechnological explorations of extremophiles. Current opinion in biotechnology, 17(3), pp.250-255. 24. Rastogi, R.P., Sinha, R.P., Singh, S.P. and Häder, D.P., 2010. Photoprotective compounds from marine organisms. Journal of industrial microbiology & biotechnology, 37(6), pp.537-558. 25. Rastogi, R.P. and Sinha, R.P., 2009. Biotechnological and industrial significance of cyanobacterial secondary metabolites. Biotechnology advances, 27(4), pp.521-539. 26. Santos, A.L., Oliveira, V., Baptista, I., Henriques, I., Gomes, N.C., Almeida, A., Correia, A. and Cunha, Â., 2013. Wavelength dependence of biological damage induced by UV radiation on bacteria. Archives of microbiology, 195(1), pp.63-74. 27. Singh, O.V. and Gabani, P., 2011. Extremophiles: radiation resistance microbial reserves and therapeutic implications. Journal of applied microbiology, 110(4), pp.851- 861. 28. Slade, D. and Radman, M., 2011. Oxidative stress resistance in Deinococcus radiodurans. Microbiology and Molecular Biology Reviews, 75(1), pp.133191. 29. Tapias, A., Leplat, C. and Confalonieri, F., 2009. Recovery of ionizingradiation damage after high doses of gamma ray in the hyperthermophilic archaeon Thermococcus gammatolerans. Extremophiles, 13(2), pp.333-343. 30. Terato, H., Suzuki, K., Nishioka, N., Okamoto, A., Shimazaki-Tokuyama, Y., Inoue, Y. and Saito, T., 2011. Characterization and radio-resistant function of manganese superoxide dismutase of Rubrobacter radiotolerans. Journal of radiation research, 52(6), pp.735-742. 31. Tian, B., Sun, Z., Shen, S., Wang, H., Jiao, J., Wang, L., Hu, Y. and Hua, Y., 2009. Effects of carotenoids from Deinococcus radiodurans on protein oxidation. Letters in applied microbiology, 49(6), pp.689-694. 32. Upadhyay, S.N., Dwarakanath, B.S., Ravindranath, T. and Mathew, T.L., 2005. Chemical radioprotectors. Defence Science Journal, 55(4), p.403. 33. Waditee-Sirisattha, R., Kageyama, H. and Takabe, T., 2016. Halophilic microorganism resources and their applications in industrial and environmental biotechnology. AIMS Microbiol, 2, pp.42-54.

Page 80

34. Yu, L.Z.H., Luo, X.S., Liu, M. and Huang, Q., 2015. Diversity of ionizing radiation resistant bacteria obtained from the Taklimakan Desert. Journal of basic microbiology, 55(1), pp.135-140. 35. Schulze-Makuch, D., Schulze-Makuch, A. and Houtkooper, J.M., 2015. The Physical, Chemical and Physiological Limits of Life. Life, 5(3), pp.14721486.

Page 81

2. Literature Review

2.1. Extremophiles

Diverse physical conditions give rise to diverse environment, where living organisms thrive. This environment can be either moderate or extreme. Temperature range of 20-40°C, pH near to neutral, atmospheric pressure of 1 atmospheric and sufficient level of water, nutrients and salts is normally considered as moderate environment. On contarary to moderate environment, there lies hostile and unusual environment i.e. hot springs, deserts, acidic and saline environment. Microbes that have the ability to persist in geochemically extreme ecological surroundings are called extremophiles. They adapt theirselves to these harsh conditions. Extremophiles can even be isolated from stratosphere and troposphere where temperature range of -20 to -40°C exists (Shivaji et al., 2004). Similarly, , and possess the ability to survive and replicate at relatively hot, acidic and alkaline conditions. Barophiles require high pressure for their sustainibility and can utilize low and high organic matter as a substrate. Radio-resistant microbes can with stand an exposure to intense radiations and chemical mutagens (Satyanarayana et al., 2005). Members of extremophiles are prevalent in different genetic linages of all three domains i.e. eucarya, archea and bacteria (Averhoff and Müller 2010).

2.2. Ionizing Radiations

Energy in the form of electromagnetic waves is called radiation. The isotopes of some elements are not stable due to which they deteriorate rapidly. In the process of decay, they are transformed into another element. During conversion they discharge ionizing radiation. These ionizing radiation possess the ability to knock out electrons from their respected orbits around another atom, which in turn charges the remaining particle. As a result, an ion is formed. These radiations act as a mutagen as they interact with molecules of the cell and alters their chemical composition hence causing mutations. For example these radiations oxidize DNA which results in programmed cell death and in some cases it leads to cancer, if the process of repairs fails (Peter et al., 2012).

2.3. Types of Radiations

Ionizing radiations includes gamma rays, x–rays, radio waves and UV radiations. These radiations harm bio molecules such as proteins, DNA, RNA by causing an oxidative damage. Sunlight contains the most basic form of radiation that is the ultra violet radiation (UVR). The UVR has a wavelength of 10-400 nm (Figure 2.1). Its energies ranges

Page 82

from 3-124 eV (Hockberger, 2002). The UVR that spreads the earth’s surface is currently increased due to constant ozone layer depletion. The largest organ of the body is skin, which is directly exposed to variety of detrimental environmental factors, UVR is one of them (Barzilai, 2010). UVR penetrates into the epidermal layers where it produces reactive oxygen species (ROS), which results in single and double DNA strand breaks (Singh and Gabani, 2011). Variety of responses ranging from inflammation, immunosuppression, and gene mutation can occur. The carcinogenesis occurs by alteration of p53 gene, which in turn is altered by the induction of cyclobutane pyrimidine dimer by mutation, thus disrupting the normal cell cycle (Klein et al., 2010). Another type of ionizing radiation is gamma radiation (GR). It is not generated by the sun. The main source of nuclear decay is GR . Exposure to radiations from nuclear facilities is associated with acute impact on human health. Fever, Fatigue, weakness, dizziness, hair loss, stomach problems may occur due to prolong exposure. The prolong exposure in some cases may lead to leukemia and leucopenia (Kurnaz et al., 2007). Microbes which are able to withstand such extreme radiations are called radio-resistant or radio extremophiles (Gabani and Singh, 2013)

2.4. Diversity of Radio-resistant Microorganisms

Different organisms show resistance to radiations because of their evolutionary adaptation. In order to withstand the increased doses of radiations, these organisms certainly have established a massive radio-resistance. Besides Chernobyl, these organisms have cosmopolitan distribution. Radioactive lakes, beaches and quarries are present in Japan, Brazil, China and India. Undeniably, minor doses of background radiations fall on almost every part on Earth. In search for additional examples bacteria were discovered having comparable abilities. Billi et al., (2000) reported different species of radio-resistant microorganisms that were able to tolerate elevated levels of radiations. The preliminary listing documented the organisms from three different domains (Table 2.1). Of all the species, Chroococcidiopsis specie, Deinococcus radiodurans, Rubrobacter radiotolerans and Thermococcus gammatolerans are the well-studied (Billi et al., 2000; Tapias et al., 2009; Terrato et al., 2011).

Among the radio-resistant microbes many are spore formers. However, others growing vegetatively are not efficient radiation resistant. Several are pathogenic which are deprived of systems due to which they cannot be manipulated genetically. Enterococcus faecium and Alcaligens are pathogenic radiation resistant bacteria. Deinococcus are noticeably unusual (Daly, 2000). Among the extreme radio-resistant microorganisms, the species of Deinococcus genus predominates. In eubacteria phylogenetic tree, it is one of the oldest lineages. In 1956, first Deinococcus strain R1 was isolated. Isolation of D. radiodurans (SARK) as an air contaminant from Ontario hospital led to the discovery of second strain. From arsenic- contaminated aquifer, arsenic resistant Deinocuccus indicus was isolated (Suresh et al., 2004). Isolation of Deinococcus saxicola, Deinococcus frigens and was reported from continental Antarctica (Hirsch et al., 2004). From Sahara desert Deinococcus deserti was isolated (De Groot et al., 2005) and isolation of Deinococcus navajonensis, Deinococcus hopiensis, and Deinococcus pimensis from single soil sample of Sonoran desert (Rainey et al., 2005). In

Page 83

an approach to isolate unusual radiation resistant bacteria, a novel Deinococcus specie from contaminated tryptone glucose yeast was isolated (Shashidhar and Bandekar, 2006). High levels of gamma radiations are found to be withstand by Thermococcus littoralis and which belong to hyperthermophilic archaea (Satyanarayana et al., 2005).

On this planet, the photosynthetic prokaryotic cyanobacteria are believed to be among the most ancient organisms. They thrive in anerobic environment and have evolved different mechanisms to reists extremely high light and UV radiations and desiccation (Sorrels et al., 2009). Although the harmful effects of these radiations are immense, yet the microorganisms are able to survive under elevated levels of radiations. Under super lethal effects of UVR, Deinococcus radiodurans are capable of survival (Yun and Lee, 2009). Harmful effects of UVR are also found to be tolerated by endolithic cyanobacteria (Rastogi et al., 2010). Due to efficient DNA repair mechanisms, cyanobacterium strain Chroococcidiopsis displays resistance to ionizing radiation (Singh and Gabani, 2011). Among the hyperthermophilic archaea, radio-resistance is widely observerd. Thermococcus gammatolerans which belongs to hyperthermophilic archaea shows immence resistantance to radiations similar to that of Deinococcus radiodurans (Confalonieri and Sommer, 2011). Species of phylum Deinococcus – Thermus i.e. Deinococcus geothermalis is found to be extremely resistant against infra-red and UV radiations (Makarova et al., 2007). The organism was reported first to be isolated from geothermal springs i.e. from aerobic environment and later from deep oceanic sub surface i.e. from the environment with complete absence of oxygen (Liedert et al., 2012). Rhodanobacter and Desulfuromonas ferrireducens were found to be present in areas with high levels of radionuclides. Radio-resistant microorganisms survive under the harmful radiations and have well- organized DNA repair mechanism, due to which they are easy to yeild primary and secondary protective metabolites (Gabani and Singh, 2013).

2.5. Adaptation of UVR Microbes to UV Radiation

Ancient organisms like cyanobacteria have advanced variety of mechanisms to preserve from the harmful UV radiation. Avoidance of stress, defense against stress and repair mechanisms are the three generalized responses of radio-resistant to stress conditions

2.5.1. Avoidance of UV Stress

Limited information is present of the UV influence on cyanobacterial vertical migration, contrary to the fact that by gliding a number of cyanobacteria are motile. Avoidance of elevated solar radiations through daily vertical migrations have been reported in species of Oscillatoria and Spirulina. In Microcoleus chthonoplastes, vertical migration has shown to be induced by UV andphotosynthetically active radiation (PAR) (Bebout and Garcia-Pichel 1995). It has also been reported that by moving downward into the mat communities, high solar radiations are escaped by motile cyanobacteria

Page 84

(Quesada and Vincent, 1997). However the influence of migration on primary productivity of mats still needs to be understood completely.

2.5.2. Defense against Stress

Production of UV-absorbing compounds by cyanaobacteria is considered as one of the important mechanisms to avoid UV photodamage. Cyanobacteria and other lower organisms have been observed to posses mycosporin like aminoacids that can effectively absorb harmful UV rediations, ultimately photo-protecting them from UVR. Such amino acids like mycosporine have maximum absorption between 310 and 360 nm (Scherer et al., 1988; Karentz et al., 1991; Ehling-Schulz et al., 1997). Substituted cyclohexenones are linked to amino acids and amino alcohols in the conformation of mycosporine amino acids. The mycosporine amino acids themselves are water soluble and are supposed to be originated from shikimate pathway (FavreBonvin et al., 1987). Cyanobacteria have a number of microsporin like amino acids (MAAs). However the particular specie and the location of pigments in them determine the relative protection provided by MAAs against UV-B damage. Only 10 to 26% photons are absorbed in some cyanobacterial species by the MAAs located in the cytoplasm. This is significant yet limited protection against harmful radiations (Garcia-Pichel and Castenholz, 1993). In Nostoc commune, MAAs that are located in the extracellular glycan plays a crucial role in photo protection. Two out of the three photons are absorbed by the pigments (Böhm et al., 1995). Colonies are exposed to high solar radiations, two pigments which are UV–A/B absorber were found with absorption limits in rage 312-335 nm (Scherer et al., 1988). One was located in the extracellular glycan. Mycosporine was the first reported that is covalently joined with oligosaccharide (Peat et al., 1994; Böhm et al.,, 1995). By absorbing the harmful radiations, the pigment provides protection to the organism. Additional protection by radical quenching may be provided by 312 chromophore of one pigment which is supposed to be a MAA-glycoside (Dunlap and Yamamoto, 1995). UV-absorbing compounds have vital role in N.commune photo protection. They have ability to survive in quiescence for long time and can withstand prevalent series of rewetting and desiccation, during which the mechanism of repair are totally ineffectual (EhlingSchulz et al., 1997). Table 2.2 presents ultraviolet radiation (UVR) induced microbial therapeutic implications and their metabolic products.

Cyanobacteria possess another pigment known as Scytonemin which have UV- shielding properties. Scytonemin located in the cyanobacterial sheath with maximum absorption of 370 nm in vivo. Its molecular mass is 544 Da. It is a yellowbrownish ,dimeric pigment with a lipid soluble properties. Indolic and phenolic subunits lay the base of its structure. Condensation of tryptophan and phenylpropanoid derived subunits have a role in the formation of this pigment (Proteau et al., 1993). UV-A irradiations strongly induce its synthesis in comparison to UV-B irradiations which have limited potential. Because of aforementioned reasons it is suggested to serve as UV-A sunscreen (Garcia Pichelet al., 1992; EhlingSchulz et al., 1997). In the course of evolution different approaches were selected that contributed to an organisms radio-resistance. These involved the

Page 85

modification of cellular metabolism and detoxification of radiation byproducts. Oxidative stress protection, efficient DNA repair mechanism,repairment of DNA double strand, and helps cell recovery from the injuries induced by radiations (Confalonieri and Sommer, 2011).

Figure 2.1: Diagrammatic representation of radiations and their effects on origin of different types of extremophiles (Source: Prashant Gabani and Om V. Singh, 2013).

Table: 2.1. Presenting resistance to Ultraviolet radiation (UVR) in diversity of radioresistant micro-organisms.

Page 86

Table: 2.2. Showing the therapeutic implications along microbial metabolic products due to Ultraviolet radiation (UVR)-inductive.

Page 87

2.5.3. Oxidative stress protection

In D. radiodurans, the ROS (reactive oxygen species) scavenging system consists of anti-oxidant enzymes and non-enzymatic antioxidants. In D. radiodurans enzymatic antioxidants like catalase (CAT) and superoxide dismutase (SOD) play an crucial role in

Page 88

resistance. Markillie et al., 1999; Makarova et al., 2001). Reactive oxygen species (ROS) harmful for cells and cause damages, but some antioxidant enzymes like superoxide dismutase and catalase act as first line of defense and guard the cells against oxidative stress. They protect the cell from damage due to the mechanism is, the enzyme superoxide dismutase acts as a catalyst in the conversion of oxygen superoxide to hydrogen peroxide. Then by the enzymes catalase or peroxidase, hydrogen peroxide is transformed into water (Luis et al., 2002). Induction of three predicted superoxide dismutases and three catalases resulted in D. radiodurans after exposure to ionizing radiations (Omelchenko et al., 2005). Superoxide dismutase are particularly encoded by the aerobic organisms such as or a few anaerobes Methanosarcina of the phylum Archaea (Brioukhanov et al., 2006). In the anaerobic radio-resistant archaea such as Thermococcus gammatolerans, superoxide reductase (SOR) is in place of superoxide dismutase (Grunden et al., 2005). During the processing of ROS, superoxide reductase is oxidized and by rubredoxin it needs to be reduced. By NADPH rubredoxin oxidoreductase (NROR) , the rubredoxin is in turn re-oxidized. By the action of rubrerythin the hydrogen peroxide is converted into water (Molina-Heredia et al., 2006).

An increased amount of Mn+2 to Fe+2 ratio provides shelter against oxidative + 3+ +2 +2 stress. Through the Fenton reaction that is (H2O2 + Fe2 2OH+ Fe ) the Mn to Fe ratio provides heightened capacity to stop the synthesis of iron dependent ROS (Imlay, 2003). Mn mechanism of action is similar to the enzymes catalase and superoxide dismutase (Seib et al., 2004). It has no relation to DNA stoppage of double strand breaks. D. radiodurans was analyze by growing the organisms in defined medium deprived of Mn+2 supplements (Daly et al., 2004). Mn+2 Intracellular concentration is 300 times higher in the most radio-resistant species and that of Fe+2 is three times lower in comparison to the most sensitive species. The level of protein oxidation was shown to be inversely correlated to the Mn+2 to Fe+2 concentration ratio. With elevated Mn+2 to Fe+2 ratio, no protein oxidation was detected (Dalyet al., 2007)(Kish et al., 2009). After exposure to irradiations, a ferritin/Dps like genes expressions can be reduced by P.furiosus. The protein coded by this gene limited the production of hydroxyl radicals by chelating iron through the Fenton reaction (Williams et al., 2007). To neutralize the protein oxidization, the decrease in the iron levels could be a common approach between some radio resistant bacteria and archaeon. However, Mn to Fe ratio cannot be conclude that radio resistant mutant E.coli strains are similar to that of wild type (Harris et al., 2009).

Protection against oxidative stress is also provided by some other ions. It was found that cell survival of Halobacterium NRC-1 to ionizing radiation was enhanced, with high concentrationof NaBr (1.7) (Kish et al., 2009). The levels of protein oxidation differs among the radio-resistant D. radiodurans and that of radio-sensitive E. coli. However, the number of DNA breaks formed in their DNA are similar to each other, even if the action of ROS indirectly resulted in 80% of DNA damage (Gerard et al., 2001). As cell require protein protection for DNA repair after exposure to irradiations, this observation led Daly and coworkers to propose that a main feature of radio resistance could be protein protection (Daly et al., 2007). Recently it was also indicated that hydrophilic proteins with low complexity (LC) regions have a role in recovery of DNA damage in D. radiodurans (Kriško et al., 2010).

Page 89

2.5.4. Non-enzymatic antioxidants

Among the non-enzymatic antioxidants, stored Mn in D. radiodurans was recommended to aid the bacterium, battering against oxidative stress. This is accomplished through non-enzymic dismutation of superoxide anion radicals (Daly et al.,2004; Ghosal et al., 2005). D. radiodurans also produce an antioxidant metabolite Pyrroloquinoline-Quinone (PQQ). This metabolite is a ROS scavenger and has the rate constants analogous to other eminent antioxidants (Misra et al., 2004). It was found that Mn and PQQ protected the proteins from oxidative stress in D. radiodurans.

Among non-enzymatic antioxidants, the carotenoids (Krinsky and Johnson, 2005) which are the efficient scavengers of ROS, chiefly of singlet oxygen (Fraser et al., 2004). Carotenoids are natural pigments, normally tetra-terpenoids, including C40 hydrocarbon backbones (carotenes) and its oxygenated derivatives (xanthophyll’s). They are manufactured by microorganisms and plants. D. radiodurans also synthesize quiet large number of carotenoids (Armstrong, 1997). The bacterium possesses 13 genes, which play a role in the biosynthesis of carotenoids. The characteristic red color of the bacterium is due to the presence of these carotenoids. However, some carotenoids are colorless such as phyotene, thus a colorless mutant might synthesize some carotenoids. In the carotenoid synthesis pathway, the major product formed is the deinoxanthin. Compared to other carotenes such as lycopene and -carotene, deinoxanthin has a stronger scavenging ability on hydrogen peroxide and superoxide and in D. radiodurans. The deinoxanthin contributes to cell resistance under oxidative stress condition. Under in vitro condition, it also inhibits protein oxidation at lower concentrations than other carotenoids.

2.6. Carotenogenesis Genes and Related Biosynthetic Pathway

2.6.1. Synthesis of Phyotene

First carotenoid is the phyotene that is produce in the biosynthesis pathway of bacteria. It is colorless and is formed of two molecules of geranyl geranyl pyrophosphate (GGPP) by condensation (Armstrong et al., 1990). Another C5 compound. Isoprenoids, quinones, terpenes, sterols and carotenoids can be obtained from IPP (Ohto et al.,1999). Phyotene is formed by the action of phyotene synthase (CrtB) on GGPP. In D. radiodurans, CrtB gene (DR0862) was determine through gene mutation and carotenoid product analysis (Tian et al., 2007; Zhang et al., 2007).

Page 90

2.6.2. Lycopene Synthesis through Desaturation of Phyotene

Phyotene is transformed into lycopene through four desaturation steps. Bacterial type phyotene desaturase (CrtI) catalyze these steps. These steps can also be catalyzed by plant type phyotene (Takaichi and Mochimaru 2007). In the genomes of Deinococcus- Thermus species only, CrtI homologs have been reported. In T. thermophilus strains HB27 and HB8, the carotenoid biosynthesis gene cluster codes CrtI. This CrtI is grouped with CrtB and GGPP synthase. Additionally the function of CrtI was proved and described in D. radiodurans (Bruggemann et al., 2007; Henne et al., 2004).

2.6.3. Lycopene Cyclization

Among acyclic and cyclic carotenoids ,carotenoid biosynthesis is diverse. CrtL or CrtY type lycopene β-cyclase catalyze the cyclization of lycopene on one or both C-ends of lycopene (Armstrong et al., 1997). In D. radiodurans an asymmetrically acting lycopene β- cyclase (CrtLm DR0801) was recognized via coexpression of CrtLm with CrtEIB genes from Pantoea stewartii in E. coli (Tao et al., 2004). It was observed to be involved in the catalysis of monocyclic carotenoids production. Accumulation of lycopene occurred in D. radiodurans, due to mutation in CrtLm gene homolog. It was confirmed that, this gene (CrtLm ) codes for lycopene cyclase in the natural host (Tian et al., 2008).

2.6.4. C-end and Ring End Modification

In monocyclic or dicyclic carotenoids, modification occurs on the C-end and ring end. These modifications comprise of hydroxylation, ketolation, desaturation, acylation and glycosylation. These modifications results in emergence of different structures and types of carotenoids found among Deinococcus-Thermus. In D. radiodurans and M.ruber, the C-end modification of monocyclic carotenoids,such as the main carotenoids need carotenoid 10,20 hydratase and carotenoid 30,40desaturases in order to attain C-10,20 hydration and C-30,40 dehydrogenation of gcarotene, correspondingly. A CruF-type carotenoid 10,20-hydratase was documented in D. radiodurans and D.geothermalis (Sun et al., 2009 A), mostly raised in photosynthetic bacteria. The C-30, 40 desaturation of g- carotene derivatives cannot take place. A C-30, 40 desaturase (CrtD) was discovered in D. radiodurans (Tian et al.,2008). As in the mutant or wild type of CrtLm, the C-30, 40 desaturated lycopene products of earlier intermediates not originated, this suggested that the CrtD in D. radiodurans they are unlike the acyclic carotenoids detailed CrtDs that are found in purple bacteria and Bradyrhizobium ORS278 (Steiger et al., 2000).

Page 91

For ketolation of C-4 or C-2 on b-ring of carotenoids in D. radiodurans or M.ruber, the presence of carotenoid ketolases are necessary. In D. radiodurans and Actinomycetale, Rhodococcus erythropolis, a CrtO-type carotenoid ketolase was documented (Tao et al., 2004). In an E. coli expression system that stores -carotene, the Deinococcus CrtO have capacity of symmetric catalyze addition to two keto groups to - carotene into finally developing canthaxanthin. (Sun et al., 2009 B). In D. radiodurans, a carotenoid biosynthetic pathway was suggested. Based on the charterization of carotenoid enzymes biosynthetic as well as intermediary product analysis (Figure 2.2) (Bing and Yuejin, 2010).

Page 92

Figure 2.2: Carotenoid biosynthetic pathway (Source: Sun and Hua, et al., 2009) showing biosynthetic enzymes and intermediate product with Deinoxanthin as the final product. Bold line indicate enzymes that been confirmed in Deinococcus radiodurans. The dotted line shows enzymes that have not been identified and involved in Deinoxanthin (final product) biosynthesis from this bacterium.

2.7. Toxic Oxygen Specie Removal

2.7.1. Role of Pigments

By photo dynamically producing reactive oxygen intermediates,oxidative stress is known to be caused by UV-A and UV-B (Cunningham et al., 1985; Shibata et al., 1996). A strategy like elimination of toxic oxygen specie can be used for mechanism of defence. Studies have shown that in plants, carotenoids have a crucial role in protection against UV-B radiations (Middleton and Teramura, 1993). Carotenoids have tremendous amount of antioxidant activity as they elliminate singlet oxygen, chlorophyll triplet and prevent lipid peroxidation (Edge et al., 1997). In response to UV-A and UV-B radiations, it has been revealed that an increase in the carotenoid/Chla ratio occurs, which supports the role of carotenoid as an significant anti-oxidant specie (Paerl, 1984; Ehling-Schulz et al., 1997; Quesada and Vincent, 1997). Changes in the carotenoid patterns of cyanobacteria such as N.commune have been examined in response to UV-B irradiations. Outer membrane bound myxoxanthophyll along with echinenone were proposed to function as UV-B photoreceptors (Ehling-Schulz et al., 1997).

Page 93

2.7.2. Role of Enzymes

Level of reactive oxygen species has shown to be decreased by scavenging enzymes like peroxidases and superoxide dismutase. Ascorbate peroxidase and catalases are the scavenging enzymes known to be produced by cyanobacteria (Miyake et al., 1991). UV-B irradiations have been reported to induce the production of superoxide dismutase’s and ascorbate peroxidases in plants and in microalga. However, in cyanobacteria the significance of enzymes with radical scavenging activities needs further investigation (Lesser, 1996; Rao et al., 1996).

2.7.3. Extracellular Polysaccharides Synthesis

UV damage can also be prevented by the extracellular polysaccharide synthesis. It provides shelter against phagocytosis, desiccation and lysis by viruses have been documented to be provided by bacterial extracellular polysaccharides (EPS) (Tease and Walker, 1987; Hill et al., 1994). In cyanobacteria, the mechanism of action of EPS containing sheath is such that it creates a buffer zone between the cell and environment. It has been noted that the yielding of extracellular glycan in N. commune is stimulated by UV-B irradiations. It was shown that the in UV-B irradiated cultures the production of EPS was three times higher in comparison to that of control cultures. In effect path for the absorpable lengths of radiations are lengthier with thicker sheaths. It has been proposed that in N. commune synthesis of EPS is roused, so that UV-B absorbing oligosaccharides matrix (mycosporines) which are located in the sheaths (Ehling-Schulz et al., 1997).

2.8. Molecular Level Adaptation to UV Stress by Efficient Repair Mechanism

An improved targets synthesis mechanism or damaged targets repair of without de novo synthesis are the possible replacements to overcome the UV–damage to the targets. The mechanism of DNA repair is common in all cell types. However, in E. coli it has been extensively studied. Several phenomena like excision repair photo reactivation and repair after replication (SOS) in E. coli are involved in the recognition and repair of UV-induced photoproducts (Walker, 1985). In the course of photo reactivation, the UV- Aand blue light activate the enzyme DNA photolyase which coverts cyclobutane type pyrimidine dimers into monomers (Pang and Hays 1991). Excision repair is not dependent on light. The process involves numerous enzymes. First damaged part of the DNA is nicked, finally the process of re-synthesis fills the gaps. Photo reactivation and excision repair, both the mechanisms have been documented to be found in cyanobacteria (O'Brien and Houghton 1982; Levine and Thiel, 1987; Kooiman et al., 1990). In cyanobacteria, the Rec-A like genes have shown to complement a rec-A deletion in E. coli (Owttrim and Coleman, 1987). Increased UV-C resistance was shown by complemented

Page 94

rec-A strains. In the SOS repair mechanism, the first step is RecA protein activation by DNA damage. The SOS genes are expressed (SOS regulon) when the RecA cleaves the LexA repressor (Walker, 1985). UV-C irradiation is used in most studies associated with DNA damage repair. However recently in Pseudomonas aureginosa, the UV-A and UV-B irradiation induction of recA expression has also been reported (Kidambi et al., 1996). 2.8.1. Enhanced Degradation of Protein and Resynthesis

To overcome the damage caused by UV radiations, enhanced protein degradation and resynthesis plays a crucial role. It helps in rapid replacement of UVsensitive proteins. In Scynechocystis sp PCC6803, in response to UV-B irradiations, an increased turnover of photosystem ii (or water-plastoquinone oxidoreductase) reaction system proteins D1 and D2 was reported. The replacement of newly produced D1 and D2 with that of UV-damaged D1 and D2 in the thylakoid was investigated (Sass et al., 1997). In the degradation of UV-B damaged D1 protein, the role of specific cleavage site has also been documented (Barbato et al., 1995). In the past few years the turnover of D1 protein has been examined in detail. It has been noticed that in most of the stressed conditions it is being regulated. In response to environmental stresses, its turnover is generally adaptive response.

2.8.2. Efficient Repair of DNA Double Strand Break

It is believed that the most hazardous and tough types of DNA breaks are the ones which are in double strands of the DNA. However, hundreds of double stranded breaks are tolerated by the organisms like D. radiodurans and T. gammatolerans (Battista et al., 1999). It was shown that after being exposed to γ radiations of 6,800 Gy, the repair of DNA double strand breaks was robust and a number of fragments were joined to reconstruct an intact piece of genome when the cell were incubated up to 3 hours in a medium supplemented with numerous nutrients (Servant et al., 2007; Tapias et al., 2009).

2.8.3. Mechanism Involved in the Repair Process

The damaged copy of DNA is also kept by the cell in the mechanism involved in the repair like single strand annealing (SSA), homologous recombination, extended

Page 95

synthesis dependent strand annealing (ESDSA) (Figure 2.3). Contrary to this non homologous recombination does not require extra copy to be preserved to join two fragments (Hefferin and Tomkinson, 2005).

2.8.3.1. Homologous Recombination In the organisms like bacteria and Saccharomyces cerevisiae that is yeast, the homologous recombination is the chief pathway eleborated in the repair of DNA double- strand breaks (Lee et al., 1999; Wyman and Kanaar, 2006). This mechanism incorporates an intact homologous DNA molecule at the damaged site to reinstate the correct DNA sequence (Figure 2.3). Generation of single stranded 3´overhang for the formation of nucleoprotein filament is the first step is the breakage of DNA double strand and repair by homologous recombination. The enzymes involved in this filament formation are RadA in archaea, RecA in bacteria and Rad51 in the cell of eukaryotes (Seitz et al., 1998). Invasion of homologous overlapping fragments; followed by the DNA strands exchange nascent DNA heteroduplex extension and resulting cross over resolution are respectively the next steps involved in the homologous recombination.

2.8.3.2. Single Strand Annealing (SSA) It has been suggested that in irradiated Deinococcal cells, single strand annealing also occurs at the very beginning besides homologous recombination. Its early occurrence corresponds to the repair observed in the Deinococcal cells (Daly and Minton, 1996). To accomplish this process it is obligatory that two copies double break of like chromosome. Single stranded overhangs are produced soon after the sectioning of DNA ends in the reaction catalyzed by exonuclease. Annealing can only takes place if there are complementary sequences in the overhangs. By the process of DNA synthesis, the single strand gaps are being filled, in the re-constructed chromosome.

2.8.3.3. Extended Synthesis Dependent Strand Annealing (ESDSA) ESDSA was proposed by Zahradka et al., (2006) as an alternative to SSA model. This model of ESDSA was the justification of their findings that, in irradiated cells the DNA fragments assembly corresponds with DNA synthesis and is much higher in comparison to un-irradiated growing cultures. In the damaged cells, an enormous number of single strand DNA is present. Both the old and newly synthesized DNA segments are present in newly assembled genome. The DNA synthesis occurs as the single stranded ending of the lower fragment occupies a partially overlapping fragments. However, differing to an aforementioned model, in classical way of double stranded DNA replication, through the displacement of Dloop, generation of single stranded fragments occurs (Figure 2.4). And this is the same as that observed in the transcription process. In transcription when the RNA polymerase progresses on the DNA template, it results in the liberation of newly synthesized RNA from the transcription machinery. The extension of strand can ensue to DNA template end. Defined reconstruction of long double strand DNA intermediates is accomplished by the annealing of those newly synthesized single stranded DNA tail, which contain complementary sequences. To reform the circular chromosome, recombination of these long intermediates takes place (Slade et al., 2009). Though many enzymes play

Page 96

crucial role in the synthesis step, but in the steps like an invasion prime DNA synthesis and direct intermediates maturation of chromosome through conventional recombination requires RecA enzyme. Previously observed in D. radiodurans resistance to radiations, the ESDSA plays a key role. P. abyssi massive DNA synthesis begun after 2 hours of their exposure to gamma radiations of dose 2,500 Gy. This suggested the role of ESDSA in Archaea (Jolivet et al., 2003). However the existence of ESDSA in archaea, is yet to be studied (Confalonieri and Sommer, 2011).

2.8.3.4. Non-Homologous End Joining

In eukaryotes joining of non-homologous end is the key pathway for repair of DNA double strand breaks (Weller et al., 2002). In bacteria it has been recently classify and characterized (Shuman and Glickman, 2007). Under ionizing radiations and desiccation conditions a protein PprA, specific from the family Deinococcacea is exceedingly induced (Tanaka et al., 2004). This protein specifically binds to doublestranded DNA having strand breaks. By doing so, it constrains the degradation of DNA by the enzymatic activity of exonuclease and facilitates DNA ligation by DNA ligase enzyme (Narumi et al., 2004; Murakami et al., 2006). Increased radio sensitivity was being observed in cells lacking the protein PprA. However, in D. radiodurans DNA double-strand break through non-homologous end joining is yet to be investigated (Confalonieri and Sommer, 2011).

Figure 2.3: Different routes for DNA break on repair figure (source: Blasius et al., 2008). During DNA both strand break repair, green color strand demonstrates newly synthesized

Page 97

DNA. Red and black color strands are showing two copies belonging to same chromosomal region.

Figure 2.4: Two steps of genome reconstitution in Deinococcus radiodurans (Cox, Keck and Battista, 2010). Once the DNA is degraded by several stress mechanisms.

Nucleases activity yields 3′-OH single stranded tails (may be 100 bp of longs). The 3′OH end search for the homologous strand, the process is mediated by recombination protein. The strand form a loop called D or displacement loop (extended synthesisdependent single-strand annealing (ESDSA). The complementary strand act as a template to for DNA synthesis. Replication bubble dissociates and hetero-duplex formation occurs, now the newly form strand with 3′-OH end serve as a template for newly synthesized strands and the remaining gaps are filled by ligases.

2.8.3.5. DNA Ends Processing All of the three foremost pathways that are involved in the DNA double-strand break repair require single stranded DNA tails in both bacteria as well as in archaea (Figure 2.5). Variety of helicases and nuclease are involved in this process (Yeeles and

Page 98

Dillingham, 2010). In the DNA ends re-sectioning and in the loading of RecA recombinase on SSB covered single stranded DNA tails, the key role is being performed by E .coli RecBCD complex and with their helicases, ATP-dependent nucleases. In E. coli, under certain circumstances if the RecBCD pathway is inactivated, an alternate pathway becomes functional that supports the repair of recombinational DNA double strand break (Horii and Clark, 1973; Mahdi and Lloyd, 1989; Sakai and Cox, 2009). To resect DNA duplex, the E. coli RecF requires the RecQ helicase and RecJ exonuclease. And in order to load RecA on SSB covered single stranded DNA, RecF requires RecO and RecR (Handa et al., 2009).

Organisms like D. radiodurans, D. geothermalis and D. deserti don’t have ability to synthesized RecB and RecC enzymes. These organisms encode all the RecF alternative pathway components. ESDSA type mechanism is observed in D.radiodurans for the repair of DNA double-strand breaks .RecFOR protein plays effective role in repair process and RecJ protenin facilitates cell’s vitality (Bentchikou et al., 2010). Moreover it was reported that cells were resistant to γ-irradiation even if they were lacking a very crucial helicase that is the RecQ required in repair process involving the RecF pathway of the E. coli. In addition to this, these cells presented a wild type repairing capability of DNA double- strand break. Contrary to this, the UvrD mutants exhibited strikingly low level of radio- resistance and the fragment assembly was also at much slower rate which indicates role of UvrD in the double stranded DNA ends processing.

Despite the close association of archea and bacteria, archea’s metabolic machinery [DNA replication, transcription and translation, repair and recombination of DNA] is more closely related to eukaryotes. For the repair of DNA double-strand breaks, eukaryotic cells use two chief pathways that is the homologous recombination and non- homologous end joining. Both of these pathways are dependent on the cell cycle phase as well as on the nature of DNA ends. To produce recombinogenic single stranded tails, the ―licensing‖ of DNA ends is the central step on which the pathway choice is being made. The key regulators of DNA end processing are the conserved complexes MRX/MRN comprising of protein Rad50, Mre11 and Xrs2/Nbs1 [in yeast or human] (Mimitou and Symington 2009). Proteins homologous to Rad50 and Mre11 are coded by archaea, however, it lacks proteins similar to that of Xrs2/Nbs1. Rad50 and Mre11 in combination with two other genes that code for a nuclease [NurA] and a helicase [HerA/MlaA] form a well conserved operon in the archaea that occurs in hyperthermophilic regions (Constantinesco et al., 2002; Constantinesco et al., 2004; Manzan et al., 2004).

In vitro studies showed that the Pyrococcus furiosus proteins NurA and HerA form a complex that’s displayed helicase or nuclease activity. They also reported this process was the results of stimulation in Rad50/Mre11 presence (Hopkins and Paull, 2008). Short 3´ ends are being produced by Rad50/Mre11 complex before enlistment of NurA/HerA complex. This leads to degradation of DNA at 5´ prime end and results in the generation of 3´ protruding ends. These ends produced may be overrun by RadA. In vitro studies involving Sulfholobous acidocaldarious have shown that exposure of cells to the 1000 Gy gamma rays dose resulted in Mre11 recruitment on DNA (Quaiser et al., 2008).

Page 99

The study further revealed the interaction of HerA, Rad50 and Mre11 in formation of complex on chromosome and their possible role in DNA repair mechanism. However, NurA was neither detected on chromosome nor was found to form a complex with Rad50/Mre11/HerA after the ionizing radiations. To explore the in vivo role of NurA in the DNA repair process in thermophilic archaea further research is required. In a polyploid archaeoan, Haloferax volcanii, which does not encode NurA and HerA proteins, the Rad50 and Mre11 roles were also investigated in vivo (Delmas et al., 2009). Dissimilar to yeast Saccharomyces cerevisiae, higher resistance to DNA damaging agents was shown by this archaeoan on deletion of rad50 and mre11. In H.volcanii cells that lack the Rad50 and Mre11 proteins, for the repair of DNA double strand breaks, homologous recombination act as prime pathway. Whereas in wild type it’s not the same. In wild type it is similar to that of mammalian cells in which NHEJ mainly perform the repair of DNA double strand break. However, for the cells to remain viable after DNA damaging treatment, the presence of RadA proteins is necessary (Delmas et al., 2009). After the reassembling of most of the particles through NHEJ process, the role of RadA –dependent HR would be late in the restoration of an intact circular chromosome. The complete set of archaeal protein involved in the NHEJ pathway still needs to be identified. Similar to that of bacteria, to favor homologous recombination, eukaryotic cells also requires accessory proteins. In irradiated cells it was observed that Rad52 and Rad55/57 facilitated the loading of the Rad51 recombinase on DNA single-stranded tails, covered by the SSB like complex RPA.

The presence of a motor protein Rad54 that translocate along the doublestranded DNA has been studied. The function of this motor protein is the promotion of chromatin remodeling along with strong stimulation of Rad51 DNA strand exchange activity. In yeast S. cerevisiae, the rad51, rad52 and rad54 are among the most ionizing radiation sensitive single mutant (Mazin et al., 2010). In archaea to support homologous recombination, proteins form by sequence homology with RadA act as accessory proteins. These accessory proteins comprise of RadA2, RadB, RadC, KaiC /aRadC and Sms proteins (Haldenby et al., 2009). In H. volcanii, reduction of cell growth, increased sensitivity to UV, and defects related to recombination are associated with the deletion of RadB. In P. furiosus, in vitro studies of RadB showed that this protein possess the ability to interact with DNA, however, it lacks the capability to catalyze the DNA strands exchange that is required for homologous recombination. To compensate for this other homologs of the eukaryotes such as Rad54 are present, which facilitates the process of homologous recombination. SsoRad54 from Sulfolobous solfataricus was suggested to be a translocase as it was able to interact with the DNA double strand under in vitro conditions. It was also revealed that SsoRad54 stimulates DNA strand exchange by interacting with the RadA protein (Haseltine and Kowalczykowski, 2009).

Page 100

Figure 2.5: Main pathways involved in the processing of DNA ends. For detail see the text.

2.9. Structure of Nucleoid

In bacteria condensed structures are formed by the combination of chromosome with proteins termed as nucleoids. High degree of genome condensation was observed in the nucleoids radio resistant genera Deinococcus and Rubrobacter. Moreover their nucleoids remained unaltered even after exposure to high dose of gamma radiations (Levin-Zaidman et al., 2003; Zimmerman and Battista, 2005). In order to efficiently repair radiation induced multiple DNA double stranded breaks, restricted DNA ends diffusion could be a possible repair mechanism. In D. radiodurans, it was also anticipated that four to ten copies of genome was prearranged (Minton, 1994). This pre-alignment helped effective error free repair [homologous recombination and ESDSA] of DNA double strand break by assisting the search for homology. Nevertheless the pre arrangement of the homologous chromosome copies is yet to be demonstrated . In E. coli large amount of histone like protein HU was present, and it was linked with the bacterial nucleoid. This HU effect the survival of cell after exposure to the gamma radiations (Boubrik and Rouviere,

1995). In D. radiodurans HU protein plays a crucial role by maintaining cell’s viability. It was shown that when this protein was expressed on temperature sensitive plasmid,

Page 101

progressive cellular depletion of HU at critical temperature range resulted in nucleoid de- condensation and fractionation, earlier to lysis of cell. These observations indicated that in D. radiodurans, HU proteins play a key important role in structure of nucleoid (Nguyen et al, 2009).

Pyrococcus and Thermococcus archaea which belongs to Euryarchaeota lineage, and also the methanogenic archaea possess histone like proteins. Their histones like proteins show structural homology to those present in the eukaryotes (Sandman and Reeve, 2006). Archaeal nucleosome is different from eukaryotic one, as it does not exist in octamer form. The archaeal histones like protein form dimers, which in turn join to form tetramer which eventually binds to the DNA. In Euryarchaea species as well as in methanogenic archaea, small proteins have been characterizes that may have a role in chromosome compaction (Pavlov et al., 2002). Sulfolobous a specie form lineage, differs in the structure of chromatin from that of found in Pyrococcus and Thermococcus. And until now there is been no specie of them, that encodes histones. AlbA and Sul7d are the two other DNA binding proteins that is used by these organisms to achieve DNA compaction (White and Bell 2002). In eukaryotes, alterations in the chromatin architecture directs the lesions to be easily accessed by DNA repair machinery. Chromatin modulation regulates he process of of transcription which is catalysed by, two significant enzymes histone acetylases and histone acetylases display a key important role (Hager et al., 2009). Similar to that of eukaryotic organisms, in archaea have adjusted the chromatin compaction. In Sulfolobous the enzymes Pat and Sir2 through the process of acetylation and deacetylation, modulate the DNA binding affinity of AlbA (Bell et al., 2002; Marshet al., 2005). As a result of deacetylation of AlbA, the repression of transcription occurs. Due to lack of complete data on global structure on archaeal genome during DNA repair, the role of nucleoid structure in radio resistance of archaea still needs to be answered (Confalonieri and Sommer, 2011).

2.10. Biotechnological Applications of UVR Microbes

2.10.1. Therapeutic Applications

Extremolytes are special organic natural compounds of extremophiles with great biotechnological potentials. They are indeed reserves of microbial metabolic processes. They do not take part in the growth, development or reproduction of the organism, yet their lack of presence results in the long-term damage to organism’s survivability, productivity and aesthetics (Figure 2.6 shows a screening strategy of novel bio-active compounds from radio-resistant microbes). The industrial significance of these extremolytes is being widely explored; however, their therapeutic implications are yet to be discovered. It is due to the presence of unique metabolism, to sustain under extreme environmental conditions and also a major cause of their diversity (Thomas and Dieckmann, 2002; Ferrer et al., 2007; Gostinčar et al., 2010). Advances in the analytical tools in the discipline of genomics, proteomics, and metabolomics have made it possible to identify the genes and proteins that may contribute in the regulation of the

Page 102

extremolytes under the extreme environment (Singh 2006; Ferrer et al., 2007; Hammon et al., 2009; Singh, Klisch et al., 2010). In search for various anti-cancerous agents,exciting developments have been made by investigating the marine organisms inhabiting the extreme conditions (Singh and Gabani 2011). Some of them are discussed below:-

2.10.2. Role of Carotenoids in Circumventing Diseases

Carotenoids are assumed to yeild certain health advantages. Reduction in the rate of numerous illness is also attributed to the presence of carotenoids. They do so by protecting cells and organisms from damaging substances (Vertuani et al., 2004). Several studies have indicated the protective role of Deinoxanthin in avoidance of certain diseases. These include eye diseases and certain types of cancers (Krinsky et al., 2005). The principal mechanism behind their ability to lessen the rate of diseases is the scavenging ability of ROS(Fan et al., 2012).

Among the number of degradation products made as a result of lipid peroxidation, malondialdehyde (MDA) is also one of the degradative products being produced (Girotti et al., 1998). It causes cell membrane disruption and also damages the DNA. The chain reactions elaborate in lipid peroxidation are inhibited by the cells via scavenging of free radicals. Thus scavenging of free radicals is one of the main anti- oxidative mechanisms involved in protection. In an in vitro study showed that carotenoid rich extracts from D. radiodurans possess strong ferric ion reducing ability and lipid peroxidation inhibition capability. It was also investigated that pre-treatment with deinoxanthin rich extract from Deinococcus radiodurans (EDR), reduced the elevated levels of AST, ALT and ALP in serum. Thus indicating the hepatoprotective and therapeutic capabilities of deinoxanthin rich extracts of D. radiodurans (Cheng et al., 2014).

Page 103

Figure 2.6: Screening strategy of bio-active compounds from extremophiles. In general bio-active compounds are of two forms: cell bound and non-cell bound. The cell bound bio-active compounds can be recovered by cell lyses and solvent extraction. While the non-cell bound compounds are excreted to the broth and can be recovered by different solvents. The second stage include of 16S rRNA sequencing genes and bio-active compound analysis through HPLC complementary methods.

2.10.3. Mycosporine Like Amino Acids

Mycosporine are photo protective compounds due their ability of maximum UV absorption. MAA absorbs UV in the range between 310-360 nm. They possess high molar

Page 104

extinction co-efficient [e=28,100-50,000 per Mcm]. Photostability, resistance to several abiotic stressors, and capability to dissipate absorbed radiations efficiently without reactive oxygen species (ROS) production are some of the properties, attributed to the presence of MAA’s (Conde et al., 2000; Whitehead and Hedges, 2005). They are colorless, small i.e. upto 400 Da, intracellular, (Singh et al., 2008). They occur in a variety of microorganisms including cyanobacteria and eukaryotic algae. Pyrimidine dimers are formed in the DNA on exposure to UVR. This dimerization leads to lethal concequences. The cell cycle may go out of control if these mutations are found in p53, which eventually leads to cancer. MAA’s are known to resist dimer formation, ultimately protecting DNA damage induced by UVR.

A new mycosporine from the lichenized ascomycete Collemacristatum, which exhibited protection against membrane destruction induced by UVB (Singh et al., 2013). Apart from this, the UVB absorbing mycosporine was also prevented pyrimidine dimer protection and was also shown to protect against erythema in cultured human keratinocytes (Singh and Gabani, 2011). Protective effects of MAA’s from harmful UVR were also observed on fibroblast cells of human skin (Torres et al., 2006; Oyamada, Kaneniwa et al., 2008). MAA’s are being used in the cosmetic industry for the formulation of UV sunscreens as they are regarded as UVR absorbers. Studies have indicated their use in the prevention of cancer such as melanoma (De la Coba et al., 2009). Prevention of sunburns, corneum stratum, malphigian thickening of dermal and hypodermal, structural and morphological changes in the biopsies of non-photoprotected skin was reported in a study in which the formulation consisted of MAA [Prophra-334 and shinorine i.e. P- 334+SH]. UV induced illness in mice skin was also prevented by the MAA formulation. Moreover, it was also reported to be associated with the maintenance of anti-oxidant defense system of the skin (De la

Coba et al., 2009). Palythine, asterina and palythinol are among the various MAA’s suggested to have a photo-protective properties.

Many microalgae species such as G. galatheanum and G. venificum are known to possess palythine (Llewellyn and Airs, 2010). Maristentorin occurs in Maristerntor dinoferus, a heterotrich ciliate. The Maristentorin has a potentially similar biological role to stentorian and blepharismim and this includes the UV irradiation defense also. For the low photo protection, Usurijene is well known. usurijene is converted to palythine by its cis-trans photo isomerization. Palythine is more photostable compared to usurijene (Mukherjee et al., 2006) (Conde et al., 2003). Despite of these compound’s promising qualities,the through therapeutic consequences of MAA’s as drug in human is quiet expected (Singh and Gabani, 2011).

2.10.4. Scytonemin

It was first reported by Nageli in 1849. He described it as yellow green pigmentation in cyanobacterial sheaths (Garcia Pichel and Castenholz, 1991). Scytonemin unique dimeric indole-phenolic structure (Proteau et al., 1993). It is exclusively produced

Page 105

by the cyanobacteria (Figure 2.7). It is considered as a true sunscreen (Cockell and Knowland, 1999)

It is found in almost 30 sheathed cyanobacterial species of various geographic sites and environments. Scytonemin absorbs UV radiations strongly with an in vivo λmax = 370 nm. Scytonemin prevents the UV-A radiations for reaching the interior of the cell by 85-90% (Proteau et al., 1993). Upto 5% of cultured cyanobacterial dry weight is constituted by this pigment and this percentage may exceed in the natural collections (Karsten 1988). Scytonemin mostly appears in the topmost layers of microbial mats. The microbial mats are thick microbial communities, found in the extreme environments and mostly grow in the areas exposed to light. In filamentous heterocystous cyanobacteria, Nostoc punctiforme, gene cluster important for the biosynthesis of Scytonemin was identified (Soule et al., 2007). A sequence of studies were carried out, in order to understand the regulation of biosynthetic gene expression (Sorrels et al., 2009; Soule et al., 2009).

Figure 2.7: Structure of Scytonemin, indolic and phenolic subunits (novel dimeric molecule) (mol. wt. 544) recognized for sheaths surrounding cyanobacteria cells.

Soule et al., (2009) recommend the cellular compartmentalization of Scytonemin biosynthesis in cyanobacteria, in a working model for UVR neutralization. In N. punctiforme, a distinctive set of eighteen gene cluster [NpR1276NpR1259] was observed. This gene cluster was involved in scytonemin biosynthesis. In the model it was proposed that trp and typ genes are induced by UVA irradiations, as a result of which tryptophan and p-hydroxyl phenyl pyruvate monomers are produced from chorismate. Two genes aroG and aroB were shown to boost the central metabolism machinery. Moreover these genes also controlled the rate limited enzymes. Whereas, in the cytoplasm certain precursors were also suggested to be processed by ScyA, ScyB, ScyC, and NpR1259. These intermediates were then transported to the periplasmic space by the mechanism which is still not known. To produce reduced forms of Scytonemin, these intermediates were subjected to different reactions by the enzymes in the periplasmic space. The periplasmic enzymes comprised of DsbA ScyD, ScyE, ScyF and TyrP. The Scytonemin that was then secreted was auto-oxidized, having yellow-brown color (Soule et al., 2009).

Page 106

In humans it has been speculated that the oncogenes are controlled by the polo- like kinases known as ATP-competitive inhibitor [PLK’s] and bind with ATPbinding sites in response turning off their activity. Because of this reason, the deep cavities in proteinase kinases ATP-binding domains are potent targets in search for

ATP-competitive inhibitors. Out of four PLK’s, fond in human’s PLK1 has been consider as a confirmed mitotic cancer target for many years (Figure 2.8). By using flash plate screening assay, the group revealed scytonemin ability to treat hyper proliferative disorders. Because of their investigation, scytonemin was idenify as a non-specific ATP competitor (Stevenson et al., 2002). It was shown in a recent study that those cells are very sensitive to the PLK1 inhibition, in which mutant Ras acts as an oncogene. This is due the fact that in these cells the key event appears to be the mitotic stress (Luo et al., 2009). It was observed that scytonemin mediated PLK1 expression inhibition, resulted in the induction of apoptosis in cancer cell and other osteosarcoma cells (Stevenson et al., 2002; Duan et al., 2010). Owing to its anti protein kinase activity it is been proposed that scytonemin may act as an antiinflammatory and anti-proliferative, drug by providing novel pharmacphore for the development of protein kinase inhibitors (Singh and Gabani, 2011).

In the presence of scytonemin and its derivatives mediated activation of SAPK.s, the cell could possibly survive (Singh and Gabani, 2011).

2.10.5. Ecotine

It is cyclic amino acid derivative of aspartate. Ecotine chemical structure is 1, 4, 5, 6-tetrahydro-2-methyl-4-pyrimidinecarboxylic (Galinski et al., 1985). It has low molecular weight. It is neutral, non-ionic, strong water binding molecule (Galinski 1993). Ecotine occurs in halophilic bacteria. The halophilic organisms thrive in extreme environmental conditions as they can grow in intensive sun irradiations, high temperature and extreme dryness (Galinski and Trüper, 1994). In nature, by synthesizing Ecotine, these organisms protect themselves. They do so by ecotine synthesis and enrichment within the cell, in order to protect their biopolymers against dehydration due to elevated temperatures, high salt concentrations and low water activity. The ecotine synthesis is encoded by a gene cluster ectABC (Nakayama et al., 2000). Ecotine is reported to be produced for commercial usage. It is used as cosmetic additive, extensive applications as biological and enzyme preparation, in pharmaceutical companies and in other fields as well. The formation of ecotine from aspartate in halophilic organisms occurs in three steps. It can be produced from H. elongata by continuous fermentation in which through the process of microfiltration, culture broth is isolated from the biomass. The ecotine containing filtrates acts as starting materials for the purification process. The purification processes include, electro dialysis, chromatography and crystallization (Lentzen and Schwarz, 2006). Ecotine protects skin from UVA induces cell damage in a number of different ways. Ecotine protected human keratinocytes cells from damage on exposure to UVA irradiations (Buenger and Driller, 2003). In the same study, it was discovered that ecotine prevented the release of UVA-induced second messenger, activation of transcription

Page 107

factor AP-2, expression of intracellular adhesion molecule 1 (part of immunoglobulin super family) as shown in figure 2.8.

It was also found that ecotine prevented the mutation of mitochondrial DNA. From this it could be concluded that ecotine mediated mechanism plays a role in stabilization of membranes structures that leads to increase resistance to possible damages induced by UVA irradiations. Bunger et al., studied the role of ecotine in the reducing the formation of sunburns caused as result of exposure to UV radiations (Bunger et al., 2001). Ecotine of 1% pretreated Langerhans cells in an in vivo study showed substantial protective effects of ecotine on Langerhans cells which were sensitive to UV stress. The Langerhans cells induce T-cells response, thus exerts an immunoprotective effect, along with protecting the skin from potential damage of UV (Lentnez et al., 2000). By inducing eminent levels of Hsp70 exhibit the cytoprotective effects of ecotine triggered by bacterial lipopolysaccharides. Ecotine mediated UVR neutralization is beneficial in the prevention of water loss in dry atopic skin, thus ecotine can stop the aging of skin (Buommino et al., 2005; Singh et al., 2011).

2.10.6. Bacterioruberin

Rubrobacter radiotoleransis is exceptionally resistant to ionizing radiations which have lethal effects on the organisms. Its resistance to ionizing radiations is even greater to that of D. radiodurans, a well-known radio-resistant bacterium. Another red-pigmented bacterium, Halobacterium salinarium containing the bacterioruberin was also shown to be extremely resistant to UVR and hydrogen peroxide (Asgarnai et al., 2000). It was documented that the organism show intensive resistant to lethal activities of DNA damaging agents including (ionizing) radiations and UV light. This indicated a direct correlation between the prevalence of bacterioruberin and DNA repair mechanism (Shah et al., 1998). Bacterioruberin plays effective role against DNA damage by ionizing radiations hence its potential use as a therapeutic agent in humans can not be undermined (Singh et al., 2011).

2.10.7. Sphaerophorin and Pannarin

Secondary metabolites of pharmaceutical importance are produced by UVtolerant organism’s (Muller et al., 2001). Among these organisms are lichens which are symbiotic associations of fungi, algae and cyanobacteria. Secondary metabolites having antioxidant activity obtained from the lichens have been considered as sunscreens for protection against UV irradiations. Protection against intense UVR have been reported in Chilean lichens (Russo et al., 2008). Protective effects on plasmid DNA was shown by both compounds. Additional research on the molecular mechanism involved in sphaerophorin and pannarin needs to be investigated (Singh et al., 2011).

Page 108

2.10.8. Circimventing Cancer and Aging

The extreme resistance of D. radiodurans to ionizing radiations and other oxidative agents is attributed to extremely efficient protection against oxidative agents and extremely efficient DNA repair. These abilities enable them to avoid or lessening DNA, RNA and protein damage and also the oxidative stress that is closely linked with cancer and aging. Increased DNA and protein oxidation due to ROS, decrease in the strength of antioxidant defenses and DNA repair and also the deposition of end products of oxidative are the sole causes of aging and cancers (Beckmann and James 1998). Delay or prevention of aging and cancer could be accomplished by the interventions designed to prevent accumulation of DNA damage and the by the oxidized protein production. In aging and cancer research, the central point is to recognize the factors that reverse the cancer development and aging process and also the designing of acceptable therapeutic strategies. In this concern, approaches of combating oxidative stress of D. radiodurans, may open new opportunities (Slade and Radman, 2011).

Figure 2.8: Scytonemin Production with exposure of UVR. Scytonemin absorbs UVRB using as sunscreen product for survival of the cell scytonemin. Figure presents a hypothetical pathway, scytonemin downregulate stress support pathways by inhibits PLK1 resulting in cancer cells destruction via apoptosis. C. Ectoine blocking cell cycle by

Page 109

inhibiting secondary messengers induction, transcription factor (AP-2) activation, and mutations of mitochondrial DNA and eventually leading to inhibition cancer induction by UV radiation or additional severe circumstances.

2.11. Radiation Resistant Extremolytes and their Biotechnological Implications

Apart from their role in therapeutics, the radio-resistant extremolytes also possess many biotechnological applications. In the bioenergy sector, their use has not been comprehensively studied. It has been now extremely important to find an inexpensive and efficient process for the production of bioethanol through the degradation of complex carbohydrates (Gabani and Singh 2013). Two novel strains of C.cellulansUVP1 and B. pumilis UVP4 were isolated by (Gabani et al., 2012). These strains were able to withstand radiations of 1.03×106J/m2 and 1.71×105 J/m2UVC respectively, and have ability to degrade cellulose under fluctuating physical and chemical conditions i.e. under high salt content, high temperature and acidic pH was shown by both B. pumilus and C. cellulans. Extremolytes related to radio-resistance are reported to be associated in the nuclear waste bioremediation and was noticed that a Ni-Fe hydrogenase from Desulfovibrio desulfuricans reduced Tc (VIII) (Luca et al., 2001). Shewanella putrefaciens and Geobacter sulfurreducenspossess an enzyme ctype cytochrome, this enzyme has the ability to reduce soluble radioisotopes of uranium into insoluble species (Lloyd et al., 2003). A new Halomonas specie strain isolated having ablity to eliminate technetium from solution by converting it into insoluble form (Fujimoto and Morita, 2006). It was reported that Geobacter sp. and Rhodoferax ferrireducens through the enzymatic mechanism has the ability to reduce radioisotopes (Kim et al., 2012).

By further reduction, the product formed can be converted in multi-component insoluble specie (Van Hullebusch et al., 2005). When grown in the presence of uranium, tellurium, and plutonium, sulfate-reducing bacteria such as Microbacterium flavescens showed capability to yeild, siderophores, organic acids and extracellular metabolites. Apart from being reduced to insoluble forms, radio-resistant extremophiles absorb these radionuclides. Effective uranium radioisotope adsorption was showed by brown marine algae Cystoseria indica (Seyrig 2010). Other microorganisms like Citrobacter freudii and Firmicutes were also reported to possess the ability to effectively adsorb radio nucleoids. Wu et al., (2006) developed a process for the bioremediation of increased concentration of uranium radioisotopes. This method involved the addition of ethanol.

For the bioremediation of heavy metals from acidic and neutral water, radioresistant organism D. radiodurans have proven to be very effective. It was reported that D. radiodurans possess a tremendous ability to remove uranium solution, i.e. upto 70% of 1Mm input uranium solution was removed by it (Misra et al., 2012). Furthermore, D. radiodurans was also found to bio remediate phthalate esters. These esters are extensively used in production of cosmetics, plasticizers and perfumes (Liao et al., 2010). Through desired genetic engineering of the organism, the bioremediation ability can be multiplied to include even more substrates (Gabani et al., 2013).

Page 110

2.11.1. Engineering of Deinococcus radiodurans for Purpose of Bioremediation

Right after the demonstration that D. radiodurans can grow in the presence of ionizing radiations at 6000 rad/ hour as compared to most radioactive DOE (department of energy U.S) waste sites in 1997, research intended to develop D. radiodurans for the purpose of bioremediation begun (Lange et al.,1998). All reported members of Deinococcus can in fact grow at this dose rate and because of their individual properties, they are foreseen as an important contributors to this technology. Recently it was documented that expression system developed for D. radiodurans, works at the temperature range of 50°C which is the optimal temperature for Deinococcus geothermalis growth (Ferreira et al., 1997). So there is a possibility of easy transference of genetic technology being developed for D. radiodurans, into D.geothermalis(Brim et al., 2000). At the start, Deinococcus growth was doubtful, during high level prolonged irradiation exposure, as it had been documented that upon DNA damage, DNA replication ceases in D. radiodurans (Minton et al., 1996). However their growth in 137Cs (cesium 137) demonstrated that these bacteria are capable of simultaneous semi-conservative DNA replication and homologous recombination (Daly and Minton 1995). These engineered strains are being used in complex bioremediation system designing (Daly, 2000).

2.11.2. Remediation of Metals

Microorganism’s ability to toxic effects resistance the of metals is commonly linked with their ability of transformation of those metals into a reduced amount of toxic chemical states (Diels et al., 1995; Lovely, 1995). Diversity of metal resistance /reduction genes are being tested in D. radiodurans .They are being examined to determine, whether they possess ability to resist common metallic waste components along with their capacity to transform those metals. Usually at lower oxidation states, the solubility of metals is reduced. The enzymes with the ability to catalyze metal reducing functions are becoming important elements of metal immobilization approach (Stephen et al., 1999). MR-1strain of Shewanella oneidensis (formerly S.putrefaciens) is highly potent at reducing Cr (VI), U (VI) and Tc (VII) to insoluble Cr (III), U (IV), and Tc (IV) precipitates respectively. This has been subjected to whole-scale genomic sequencing. This might contribute to pattern of genes targeted for expression in D. radiodurans (Daly, 2000).

Expanding on any natural metal remediating capability that D. radiodurans has, is an alternative path to designing D. radiodurans for metal remediation. Surprisingly, in the presence of humic acid, U (VI) and Tc (VII) can be reduced by an anaerobe D.

Page 111

radiodurans). However, Cr (VI) can be decreased in the absence of humic acid. The modification and enhancement of these functions, might be possible through genetic engineering by using its genomic sequence as a lead to manipulation.

In DOE wastes, lead, chromium and mercury are the most ubiquitious contaminants of heavy metals. A series of genetic vectors being analyze in D. radiodurans that encode resistance to these metals i.e. Escherichia coli (Summers 1986), highly characterized mer A locus has been cloned into D. radiodurans. Mercuric ion reductase (Mer A) is encoded by mer A. Highly toxic thiol reactive mercuric ion Hg (II) is reduced by this enzyme, to much less nearly inert elemental and volatile Hg (0). To regulate mer A expression, by changing its cellular gene dosage, four different D. radiodurans expression systems were developed (Brim et al., 2000). Briefly, the Mer A expressing strains of D. radiodurans shows resistant to the bactericidal effects of ionic Hg (II), at concentrations (30-50µM), well above the highest concentration recorded for mercury contaminated DOE wastes sites (10µM). The strains have capability to reduce toxic substances like, Hg (II) to Hg (0) proficiently. Other metal reducing/resistance activity that are specific to remediate metal ions, have been cloned into D. radiodurans are being examined include genes from Desulfovibrio vulgaris (cytc3), U (VI), Ralstonia eutrophus CH34 (czc) Cd(II), Co (II), ), Zn (II), and Bacillus thuringiensis Cr (VI) (Daly, 2000).

2.12. Conclusion

The intention of this article has been to explore the radio-resistant microbes, effect of UV on their genome with repair mechanisms. The correlation of different metal ions in correlation to their survival in presence of high UV and gamma radiation doses. The complex physiology and other biology based approaches i.e. genomics proteomic and metabolomics can make these microbes potential candidates for development of effective therapeutic agents. Obstacles must be overcome to utilize UVR resistant microbes for the removal of toxic heavy metals left from the cold war. These extremophiles can also be utilized for uranium bioremediation as they possess the ability to reduce soluble radioisotopes of uranium into insoluble species. Various extremolytes and other active enzymes are thought to be produced due to high UV stress that can be used as biopharmaceutical products i.e anti-cancerous, anticholestrolic and antidiabetic drugs and other for biodegradation of toxic radioactive compounds in different nuclear wastes. There are various limiting factors for commercialization of these extremolytes and extremozymes from UVR microbes. Increase research efforts need to be made via utilizing Response Surface Methodology (RSM) approach of optimization and screening, the bioactive compounds from these extremophiles, metabolic engineering could allow the potent mass production.

References

1. Armstrong, G.A., 1997. Genetics of eubacterial carotenoid biosynthesis: a colorful tale. Annual Reviews in Microbiology, 51(1), pp.629-659.

Page 112

2. Armstrong, G.A., Alberti, M. and Hearst, J.E., 1990. Conserved enzymes mediate the early reactions of carotenoid biosynthesis in nonphotosynthetic and photosynthetic prokaryotes. Proceedings of the National Academy of Sciences, 87(24), pp.9975-9979. 3. Asgarani, E., Terato, H., Asagoshi, K., Shahmohammadi, H.R., Ohyama, Y., Saito, T., Yamamoto, O. and Ide, H., 2000. Purification and characterization of a novel DNA repair enzyme from the extremely radioresistant bacterium Rubrobacter radiotolerans. Journal of radiation research, 41(1), pp.19-34. 4. Averhoff, B. and Müller, V., 2010. Exploring research frontiers in microbiology: recent advances in halophilic and thermophilic extremophiles. Research in microbiology, 161(6), pp.506-514. 5. Barbato, R., Frizzo, A., Friso, G., Rigoni, F. and Giacometti, G.M., 1995. Degradation of the D1 Protein of Photosystem II Reaction Centre by Ultraviolet B Radiation Requires the Presence of Functional Manganese on the Donor Side. European Journal of Biochemistry, 227(3), pp.723-729. 6. Barzilai, A., 2010. DNA damage, neuronal and glial cell death and neurodegeneration. Apoptosis, 15(11), pp.1371-1381. 7. Battista, J.R., Earl, A.M. and Park, M.J., 1999. Why is Deinococcus radiodurans so resistant to ionizing radiation?. Trends in microbiology, 7(9), pp.362-365. 8. Bebout, B.M. and Garcia-Pichel, F., 1995. UV B-induced vertical migrations of cyanobacteria in a microbial mat. Applied and Environmental Microbiology, 61(12), pp.4215-4222. 9. Beckman, K.B. and Ames, B.N., 1998. The free radical theory of aging matures. Physiological reviews, 78(2), pp.547-581. 10. Bell SD, Botting CH et al (2002) The interaction of Alba, a conserved archaeal chromatin protein, with Sir2 and its regulation by acetylation. Science 296(5565): 148-151. 11. Bentchikou, E., Servant, P., Coste, G. and Sommer, S., 2010. A major role of the RecFOR pathway in DNA double-strand-break repair through ESDSA in Deinococcus radiodurans. PLoS Genet, 6(1), p.e1000774. 12. Billi, D., Friedmann, E.I., Hofer, K.G., Caiola, M.G. and OcampoFriedmann, R., 2000. Ionizing-radiation resistance in the desiccation-tolerant cyanobacterium Chroococcidiopsis. Applied and Environmental

Page 113

Microbiology, 66(4), pp.1489-1492. 13. Bing, T., Yuejin, H., 2010. Carotenoid biosynthesis in extremophilic Deinococcus thermus bacteria.Trends Microbiol 18(11): 512-520. 14. Böhm, G.A., Pfleiderer, W., Böger, P. and Scherer, S., 1995. Structure of a novel oligosaccharide-mycosporine-amino acid ultraviolet A/B sunscreen pigment from the terrestrial cyanobacterium Nostoc commune. Journal of Biological Chemistry, 270(15), pp.8536-8539. 15. Boubrik, F. and Rouviere-Yaniv, J., 1995. Increased sensitivity to gamma irradiation in bacteria lacking protein HU. Proceedings of the National Academy of Sciences, 92(9), pp.3958-3962. 16. Brim, H., McFarlan, S.C., Fredrickson, J.K., Minton, K.W., Zhai, M., Wackett, L.P. and Daly, M.J., 2000. Engineering Deinococcus radiodurans for metal remediation in radioactive mixed waste environments. Nature biotechnology, 18(1), pp.85-90. 17. Brioukhanov, A.L., Netrusov, A.I. and Eggen, R.I., 2006. The catalase and superoxide dismutase genes are transcriptionally up-regulated upon oxidative stress in the strictly anaerobic archaeon Methanosarcina barkeri. Microbiology, 152(6), pp.1671-1677. 18. Brüggemann, H. and Chen, C., 2006. Comparative genomics of : plasticity of the megaplasmid and its contribution to a thermophilic lifestyle. Journal of Biotechnology, 124(4), pp.654-661. 19. Buenger, J., Driller, H., (2003) Ectoin: an effective natural substance to prevent UVA-induced premature photoaging. Skin Pharmacol Physiol 17(5): 232-237. 20. Carignan, M.O., Cardozo, K.H., Oliveira-Silva, D., Colepicolo, P. and Carreto, J.I., 2009. Palythine–threonine, a major novel mycosporine-like amino acid (MAA) isolated from the hermatypic coral Pocillopora capitata. Journal of Photochemistry and Photobiology B: Biology, 94(3), pp.191-200. 21. Chikara, O.H.T.O., Ishida, C., Koike-Takeshita, A., Yokoyama, K., Muramatsu, M., Nishino, T. and Obata, S., 1999. Gene cloning and overexpression of a geranylgeranyl diphosphate synthase of an extremely thermophilic bacterium, Thermus thermophilus. Bioscience, biotechnology, and biochemistry, 63(2), pp.261- 270.

Page 114

22. Coba, F.D.L., Aguilera, J. and Figueroa, F.L., 2007. Use of mycosporine-type amino acid Porphyra-334 as an antioxidant. Intl. Patent WO2007/026035 A, 2. 23. Cockell, C.S., and Knowland, J., 1999. Ultraviolet radiation screening compounds. Biological Reviews, 74(3), pp.311-345. 24. Conde FR., Carignan MO et al (2003) In Vitro cis–trans Photoisomerization of Palythene and Usujirene. Implications on the In Vivo Transformation of Mycosporine like Amino Acids. Photochem photobiol 77(2): 146-150. 25. Conde, F.R., Churio, M.S. and Previtali, C.M., 2000. The photoprotector mechanism of mycosporine-like amino acids. Excited-state properties and photostability of porphyra-334 in aqueous solution. Journal of Photochemistry and Photobiology B: Biology, 56(2), pp.139-144. 26. Confalonieri, F. and Sommer, S., 2011. Bacterial and archaeal resistance to ionizing radiation. In Journal of Physics: Conference Series (Vol. 261, No. 1, p. 012005). IOP Publishing. 27. Constantinesco, F., Forterre, P. and Elie, C., 2002. NurA, a novel 5′–3′ nuclease gene linked to rad50 and mre11 homologs of thermophilic Archaea. EMBO reports, 3(6), pp.537-542. 28. Constantinesco, F., Forterre, P., Koonin, E.V., Aravind, L. and Elie, C., 2004. A bipolar DNA helicase gene, herA, clusters with rad50, mre11 and nurA genes in thermophilic archaea. Nucleic acids research, 32(4), pp.1439-1447. 29. Cunningham, M.L., Johnson, J.S., Giovanazzi, S.M. and Peak, M.J., 1985. Photosensitized production of superoxide anion by monochromatic (290–405 nm) ultraviolet irradiation of NADH and NADPH coenzymes. Photochemistry and photobiology, 42(2), pp.125-128. 30. Dai, J., Wang, Y., Zhang, L., Tang, Y., Luo, X., An, H. and Fang, C., 2009. Hymenobacter tibetensis sp. nov., a UV-resistant bacterium isolated from Qinghai– Tibet plateau. Systematic and applied microbiology, 32(8), pp.543-548. 31. Daly MJ, Gaidamakova E et al (2004) Accumulation of Mn (II) in Deinococcus radiodurans facilitates gamma-radiation resistance. Science 306(5698): 1025-1028. 32. Daly MJ, Gaidamakova EK et al (2007) Protein oxidation implicated as the primary determinant of bacterial . PLoS Biology 5(4): e92.

Page 115

33. Daly, M.J. and Minton, K.W., 1996. An alternative pathway of recombination of chromosomal fragments precedes recA-dependent recombination in the radioresistant bacterium Deinococcus radiodurans. Journal of Bacteriology, 178(15), pp.4461-4471. 34. Daly, M.J., 2000. Engineering radiation-resistant bacteria for environmental biotechnology. Current opinion in biotechnology, 11(3), pp.280-285. 35. De Groot, A., Chapon, V., Servant, P., Christen, R., Fischer-Le Saux, M., Sommer, S. and Heulin, T., 2005. Deinococcus deserti sp. nov., a gammaradiation- tolerant bacterium isolated from the Sahara Desert. International journal of systematic and evolutionary microbiology, 55(6), pp.2441-2446. 36. De la Coba, F., Aguilera, J., De Galvez, M.V., Alvarez, M., Gallego, E., Figueroa, F.L. and Herrera, E., 2009. Prevention of the ultraviolet effects on clinical and histopathological changes, as well as the heat shock protein-70 expression in mouse skin by topical application of algal UV-absorbing compounds. Journal of dermatological science, 55(3), pp.161-169. 37. De la Coba, F., Aguilera, J., Figueroa, F.L., De Gálvez, M.V. and Herrera, E., 2009. Antioxidant activity of mycosporine-like amino acids isolated from three red macroalgae and one marine lichen. Journal of Applied Phycology, 21(2), pp.161-169. 38. Delmas, S., Shunburne, L., Ngo, H.P. and Allers, T., 2009. Mre11-Rad50 promotes rapid repair of DNA damage in the polyploid archaeon Haloferax volcanii by restraining homologous recombination. PLoS Genet, 5(7), p.e1000552. 39. Diels, L., Dong, Q., van der Lelie, D., Baeyens, W. and Mergeay, M., 1995. Theczc operon ofAlcaligenes eutrophus CH34: from resistance mechanism to the removal of heavy metals. Journal of Industrial Microbiology & Biotechnology, 14(2), pp.142- 153. 40. Duan, Z., Ji, D., Weinstein, E.J., Liu, X., Susa, M., Choy, E., Yang, C., Mankin, H. and Hornicek, F.J., 2010. Lentiviral shRNA screen of human kinases identifies PLK1 as a potential therapeutic target for osteosarcoma. Cancer letters, 293(2), pp.220-229. 41. Dunlap, W.C. and Yamamoto, Y., 1995. Small-molecule antioxidants in marine organisms: antioxidant activity of mycosporine-glycine. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular

Page 116

Biology, 112(1), pp.105-114. 42. Edge, R., McGarvey, D.J. and Truscott, T.G., 1997. The carotenoids as antioxidants—a review. Journal of Photochemistry and Photobiology B: Biology, 41(3), pp.189-200. 43. Ehling-Schulz, M., Bilger, W. and Scherer, S., 1997. UV-B-induced synthesis of photoprotective pigments and extracellular polysaccharides in the terrestrial cyanobacterium Nostoc commune. Journal of Bacteriology, 179(6), pp.19401945. 44. Eker, A.P., Kooiman, P., Hessels, J.K. and Yasui, A., 1990. DNA photoreactivating enzyme from the cyanobacterium Anacystis nidulans. Journal of Biological Chemistry, 265(14), pp.8009-8015. 45. Fan, L., Li, J., Deng, K. and Ai, L., 2012. Effects of drying methods on the antioxidant activities of polysaccharides extracted from Ganoderma lucidum. Carbohydrate Polymers, 87(2), pp.1849-1854. 46. Favre-Bonvin, J., Bernillon, J., Salin, N. and Arpin, N., 1987. Biosynthesis of mycosporines: mycosporine glutaminol in Trichothecium roseum. Phytochemistry, 26(9), pp.2509-2514. 47. Ferreira, A.C., Nobre, M.F., Rainey, F.A., Silva, M.T., Wait, R., Burghardt, J., Chung, A.P. and Da Costa, M.S., 1997. Deinococcus geothermalis sp. nov. and Deinococcus murrayi sp. nov., two extremely radiation-resistant and slightly thermophilic species from hot springs. International Journal of Systematic and Evolutionary Microbiology, 47(4), pp.939-947. 48. Ferrer, M., Golyshina, O., Beloqui, A. and Golyshin, P.N., 2007. Mining enzymes from extreme environments. Current opinion in microbiology, 10(3), pp.207-214. 49. Flores, M.R., Ordoñez, O.F., Maldonado, M.J. and Farías, M.E., 2009. Isolation of UV-B resistant bacteria from two high altitude Andean lakes (4,400 m) with saline and non saline conditions. The Journal of general and applied microbiology, 55(6), pp.447-458. 50. Fraser, P.D. and Bramley, P.M., 2004. The biosynthesis and nutritional uses of carotenoids. Progress in lipid research, 43(3), pp.228-265. 51. Fujimoto, K. and Morita, T., 2006. Aerobic removal of technetium by a marine Halomonas strain. Applied and environmental microbiology, 72(12), pp.7922-7924.

Page 117

52. Gabani, P. and Singh, O.V., 2013. Radiation-resistant extremophiles and their potential in biotechnology and therapeutics. Applied microbiology and biotechnology, 97(3), pp.993-1004. 53. Gabani, P. and Singh, O.V., 2013. Radiation-resistant extremophiles and their potential in biotechnology and therapeutics. Applied microbiology and biotechnology, 97(3), pp.993-1004. 54. Gabani, P. and Singh, O.V., 2013. Radiation-resistant extremophiles and their potential in biotechnology and therapeutics. Applied microbiology and biotechnology, 97(3), pp.993-1004. 55. Gabani, P., Copeland, E., Chandel, A.K. and Singh, O.V., 2012. Ultraviolet radiation resistant isolates revealed cellulose degrading species of Cellulosimicrobium cellulans (UVP1) and Bacillus pumilus (UVP4). Biotechnology and applied biochemistry, 59(5), pp.395-404. 56. Galinski, E.A. and Trüper, H.G., 1994. Microbial behaviour in salt-stressed ecosystems. FEMS Microbiology Reviews, 15(2-3), pp.95-108. 57. Galinski, E.A., 1993. Compatible solutes of halophilic eubacteria: molecular principles, water-solute interaction, stress protection. Experientia, 49(6-7), pp.487- 496. 58. Galinski, E.A., PFEIFFER, H.P. and Trueper, H.G., 1985. 1, 4, 5, 6 Tetrahydro 2 methyl 4 pyrimidinecarboxylic acid. European Journal of Biochemistry, 149(1), pp.135-139. 59. Garcia Pichel, F. and Castenholz, R.W., 1991. Characterization and biological implications of scytonemin, a cyanobacterial sheath pigment. Journal of Phycology, 27(3), pp.395-409. 60. Garcia-Pichel, F. and Castenholz, R.W., 1993. Occurrence of UV-absorbing, mycosporine-like compounds among cyanobacterial isolates and an estimate of their screening capacity. Applied and Environmental Microbiology, 59(1), pp.163-169. 61. Garcia Pichel, F., Sherry, N.D. and Castenholz, R.W., 1992. Evidence for an ultraviolet sunscreen role of the extracellular pigment scytonemin in the terrestrial cyanobacterium Chiorogloeopsis sp. Photochemistry and Photobiology, 56(1), pp.17- 23. 62. Gerard, E., Jolivet, E., Prieur, D. and Forterre, P., 2001. DNA protection mechanisms are not involved in the radioresistance of the hyperthermophilic

Page 118

archaea Pyrococcus abyssi and P. furiosus. Molecular Genetics and Genomics, 266(1), pp.72-78. 63. Ghosal, D., Omelchenko, M.V., Gaidamakova, E.K., Matrosova, V.Y., Vasilenko, A., Venkateswaran, A., Zhai, M., Kostandarithes, H.M., Brim, H., Makarova, K.S. and Wackett, L.P., 2005. How radiation kills cells: survival of Deinococcus radiodurans and Shewanella oneidensis under oxidative stress. FEMS Microbiology Reviews, 29(2), pp.361-375. 64. Girotti, A.W., 1998. Lipid hydroperoxide generation, turnover, and effector action in biological systems. Journal of lipid research, 39(8), pp.1529-1542.

65. Gostinčar, C., Grube, M., De Hoog, S., Zalar, P. and Gunde-Cimerman, N., 2010. Extremotolerance in fungi: evolution on the edge. FEMS microbiology ecology, 71(1), pp.2-11. 66. Grunden, A.M., Jenney, F.E., Ma, K., Ji, M., Weinberg, M.V. and Adams, M.W., 2005. In vitro reconstitution of an NADPH-dependent superoxide reduction pathway from Pyrococcus furiosus. Applied and environmental microbiology, 71(3), pp.1522- 1530. 67. Hager, G.L., McNally, J.G. and Misteli, T., 2009. Transcription dynamics. Molecular cell, 35(6), pp.741-753. 68. Haldenby, S., White, M.F. and Allers, T., 2009. RecA family proteins in archaea: RadA and its cousins. 69. Hammon, J., Palanivelu, D.V., Chen, J., Patel, C. and Minor, D.L., 2009. A green fluorescent protein screen for identification of well expressed membrane proteins from a cohort of extremophilic organisms. Protein Science, 18(1), pp.121-133. 70. Handa, N., Morimatsu, K., Lovett, S.T. and Kowalczykowski, S.C., 2009. Reconstitution of initial steps of dsDNA break repair by the RecF pathway of E. coli. Genes & Development, 23(10), pp.1234-1245.

71. Harris, D.R., Pollock, S.V., Wood, E.A., Goiffon, R.J., Klingele, A.J., Cabot, E.L., Schackwitz, W., Martin, J., Eggington, J., Durfee, T.J. and Middle, C.M., 2009. Directed evolution of ionizing radiation resistance in Escherichia coli. Journal of Bacteriology, 191(16), pp.5240-5252.

72. Haseltine, C.A. and Kowalczykowski, S.C., 2009. An archaeal Rad54 protein remodels DNA and stimulates DNA strand exchange by RadA. Nucleic acids research, p.gkp068.

Page 119

73. Hefferin, M.L. and Tomkinson, A.E., 2005. Mechanism of DNA doublestrand break repair by non-homologous end joining. DNA repair, 4(6), pp.639648. 74. Henne, A., Brüggemann, H., Raasch, C., Wiezer, A., Hartsch, T., Liesegang, H., Johann, A., Lienard, T., Gohl, O., Martinez-Arias, R. and Jacobi, C., 2004. The genome sequence of the extreme Thermus thermophilus. Nature biotechnology, 22(5), pp.547-553. 75. Hill, D.R., Peat, A. and Potts, M., 1994. Biochemistry and structure of the glycan secreted by desiccation-tolerantNostoc commune (Cyanobacteria). Protoplasma, 182(3), pp.126-148. 76. Hirsch, P., Gallikowski, C.A., Siebert, J., Peissl, K., Kroppenstedt, R., Schumann, P., Stackebrandt, E. and Anderson, R., 2004. Deinococcus frigens sp. nov., Deinococcus saxicola sp. nov., and Deinococcus marmoris sp. nov., low temperature and draught-tolerating, UV-resistant bacteria from continental Antarctica. Systematic and applied microbiology, 27(6), pp.636-645.

77. Hockberger, P.E., 2002. A history of ultraviolet photobiology for humans, animals and microorganisms. Photochemistry and photobiology, 76(6), pp.561579. 78. Holembiovs' ka, S.L., Lavrinchuk, V. and Matseliukh, B.P., 2008. Resistance of the colored and non-pigmented mutants of Streptomyces globisporus 1912 to the action of UV-rays. Mikrobiolohichnyi zhurnal (Kiev, Ukraine: 1993), 70(5), pp.23-26. 79. Hopkins, B.B. and Paull, T.T., 2008. The P. furiosus mre11/rad50 complex promotes 5′ strand resection at a DNA double-strand break. Cell, 135(2), pp.250-260. 80. Horii, Z.I. and Clark, A.J., 1973. Genetic analysis of the recF pathway to genetic recombination in Escherichia coli K12: isolation and characterization of mutants. Journal of molecular biology, 80(2), pp.327IN3329-328344. 81. Imlay, J.A., 2003. Pathways of oxidative damage. Annual Reviews in Microbiology, 57(1), pp.395-418. 82. Jolivet, E., Matsunaga, F., Ishino, Y., Forterre, P., Prieur, D. and Myllykallio, H., 2003. Physiological responses of the hyperthermophilic archaeon ―Pyrococcus abyssi‖ to DNA damage caused by ionizing radiation. Journal of bacteriology, 185(13), pp.3958-3961. 83. Karentz, D., McEuen, F.S., Land, M.C. and Dunlap, W.C., 1991. Survey of mycosporine-like amino acid compounds in Antarctic marine organisms: potential protection from ultraviolet exposure. Marine Biology, 108(1), pp.157166.

Page 120

84. Karsten, U., Escoubeyrou, K. and Charles, F., 2009. The effect of redissolution solvents and HPLC columns on the analysis of mycosporine-like amino acids in the eulittoral macroalgae Prasiola crispa and Porphyra umbilicalis. Helgoland Marine Research, 63(3), p.231. 85. Karsten, U., Maier, J. and Garcia-Pichel, F., 1998. Seasonality in UVabsorbing compounds of cyanobacterial mat communities from an intertidal mangrove flat. Aquatic Microbial Ecology, 16(1), pp.37-44. 86. Kidambi, S.P., Booth, M.G., Kokjohn, T.A. and Miller, R.V., 1996. recAdependence of the response of Pseudomonas aeruginosa to UVA and UVB irradiation. Microbiology, 142(4), pp.1033-1040. 87. Kim, S.J., Koh, D.C., Park, S.J., Cha, I.T., Park, J.W., Na, J.H., Roh, Y., Ko, K.S., Kim, K. and Rhee, S.K., 2012. Molecular analysis of spatial variation of iron- reducing bacteria in riverine alluvial aquifers of the Mankyeong River. The journal of microbiology, 50(2), pp.207-217. 88. Kish, A., Kirkali, G., Robinson, C., Rosenblatt, R., Jaruga, P., Dizdaroglu, M. and DiRuggiero, J., 2009. Salt shield: intracellular salts provide cellular protection against ionizing radiation in the halophilic archaeon, Halobacterium salinarum NRC 1. Environmental microbiology, 11(5), pp.1066-1078. 89. Klein, A.M., Brash, D.E., Jones, P.H. and Simons, B.D., 2010. Stochastic fate of p53-mutant epidermal progenitor cells is tilted toward proliferation by UV B during preneoplasia. Proceedings of the National Academy of Sciences, 107(1), pp.270-275. 90. Krinsky, N.I. and Johnson, E.J., 2005. Carotenoid actions and their relation to health and disease. Molecular aspects of medicine, 26(6), pp.459-516.

91. Kriško, A., Smole, Z., Debret, G., Nikolić, N. and Radman, M., 2010. Unstructured hydrophilic sequences in prokaryotic proteomes correlate with dehydration tolerance and host association. Journal of molecular biology, 402(5), pp.775-782.

92. Kurnaz, A., Küçükömeroğlu, B., Keser, R., Okumusoglu, N.T., Korkmaz, F., Karahan, G. and Çevik, U., 2007. Determination of radioactivity levels and hazards of soil and sediment samples in Fırtına Valley (Rize, Turkey). Applied Radiation and Isotopes, 65(11), pp.1281-1289. 93. Lange, C.C., Wackett, L.P., Minton, K.W. and Daly, M.J., 1998. Engineering a recombinant Deinococcus radiodurans for organopollutant degradation in radioactive mixed waste environments. Nature biotechnology, 16(10), pp.929-933.

Page 121

94. Lecointe, F., Shevelev, I.V., Bailone, A., Sommer, S. and Hübscher, U., 2004. Involvement of an X family DNA polymerase in double stranded break repair in the radioresistant organism Deinococcus radiodurans. Molecular microbiology, 53(6), pp.1721-1730. 95. Lee, S.E., Pâques, F., Sylvan, J. and Haber, J.E., 1999. Role of yeast SIR genes and mating type in directing DNA double-strand breaks to homologous and non- homologous repair paths. Current Biology, 9(14), pp.767-770. 96. Lentzen, G. and Schwarz, T., 2006. Extremolytes: natural compounds from extremophiles for versatile applications. Applied microbiology and biotechnology, 72(4), pp.623-634. 97. Lesser, M.P., 1996. Elevated temperatures and ultraviolet radiation cause oxidative stress and inhibit photosynthesis in symbiotic dinoflagellates. Limnology and oceanography, 41(2), pp.271-283 98. Levine, E.L.A.I.N.E. and Thiel, T.E.R.E.S.A., 1987. UV-inducible DNA repair in the cyanobacteria Anabaena spp. Journal of bacteriology, 169(9), pp.3988-3993. 99. Levin-Zaidman, S., Englander, J., Shimoni, E., Sharma, A.K., Minton, K.W. and Minsky, A., 2003. Ringlike structure of the Deinococcus radiodurans genome: a key to radioresistance?. Science, 299(5604), pp.254-256. 100. Li, Y., Qian, Z.J., Ryu, B., Lee, S.H., Kim, M.M. and Kim, S.K., 2009. Chemical components and its antioxidant properties in vitro: an edible marine brown alga, Ecklonia cava. Bioorganic & Medicinal Chemistry, 17(5), pp.19631973. 101. Liao, C.S., Chen, L.C., Chen, B.S. and Lin, S.H., 2010. Bioremediation of endocrine disruptor di-n-butyl phthalate ester by Deinococcus radiodurans and Pseudomonas stutzeri. Chemosphere, 78(3), pp.342-346. 102. Liedert, C., Peltola, M., Bernhardt, J., Neubauer, P. and Salkinoja-Salonen, M., 2012. Physiology of resistant Deinococcus geothermalis bacterium aerobically cultivated in low-manganese medium. Journal of bacteriology, 194(6), pp.1552- 1561. 103. Llewellyn, C.A. and Airs, R.L., 2010. Distribution and abundance of MAAs in 33 species of microalgae across 13 classes. Marine drugs, 8(4), pp.1273-1291. 104. Lloyd, J.R., Leang, C., Myerson, A.L.H., Coppi, M.V., Cuifo, S., Methe, B., Sandler, S.J. and Lovley, D.R., 2003. Biochemical and genetic characterization

Page 122

of PpcA, a periplasmic c-type cytochrome in Geobacter sulfurreducens. Biochemical Journal, 369(1), pp.153-161. 105. Lobban, C., Schefter, M., Simpson, A., Pochon, X., Pawlowski, J. and Foissner, W., 2002. Maristentor dinoferus n. gen., n. sp., a giant heterotrich ciliate (Spirotrichea: Heterotrichida) with zooxanthellae, from coral reefs on Guam, Mariana Islands. Marine Biology, 140(2), pp.411-423. 106. Lobban, C.S., Hallam, S.J., Mukherjee, P. and Petrich, J.W., 2007. Photophysics and Multifunctionality of Hypericin Like Pigments in Heterotrich Ciliates: A Phylogenetic Perspective. Photochemistry and photobiology, 83(5), pp.1074-1094. 107. Louis, P., Trüper, H.G. and Galinski, E.A., 1994. Survival of Escherichia coli during drying and storage in the presence of compatible solutes. Applied microbiology and biotechnology, 41(6), pp.684-688. 108. Lovley, D.R., 1995. Bioremediation of organic and metal contaminants with dissimilatory metal reduction. Journal of Industrial Microbiology & Biotechnology, 14(2), pp.85-93. 109. Luis, A., Corpas, F.J., Sandalio, L.M., Palma, J.M., Gómez, M. and Barroso, J.B., 2002. Reactive oxygen species, antioxidant systems and nitric oxide in peroxisomes. Journal of Experimental Botany, 53(372), pp.1255-1272. 110. Luo, J., Emanuele, M.J., Li, D., Creighton, C.J., Schlabach, M.R., Westbrook, T.F., Wong, K.K. and Elledge, S.J., 2009. A genome-wide RNAi screen identifies multiple synthetic lethal interactions with the Ras oncogene. Cell, 137(5), pp.835- 848. 111. Mahdi, A.A. and Lloyd, R.G., 1989. Identification of the recR locus of Escherichia coli K-12 and analysis of its role in recombination and DNA repair. Molecular and General Genetics MGG, 216(2-3), pp.503-510. 112. Makarova, K.S., Aravind, L., Wolf, Y.I., Tatusov, R.L., Minton, K.W., Koonin, E.V. and Daly, M.J., 2001. Genome of the extremely radiationresistant bacterium Deinococcus radiodurans viewed from the perspective of comparative genomics. Microbiology and Molecular Biology Reviews, 65(1), pp.44-79. 113. Makarova, K.S., Omelchenko, M.V., Gaidamakova, E.K., Matrosova, V.Y., Vasilenko, A., Zhai, M., Lapidus, A., Copeland, A., Kim, E., Land, M. and Mavromatis, K., 2007. Deinococcus geothermalis: the pool of extreme radiation resistance genes shrinks. PLoS One, 2(9), p.e955.

Page 123

114. Manzan, A., Pfeiffer, G., Hefferin, M.L., Lang, C.E., Carney, J.P. and Hopfner, K.P., 2004. MlaA, a hexameric ATPase linked to the Mre11 complex in archaeal genomes. EMBO reports, 5(1), pp.54-59. 115. Markillie, L.M., Varnum, S.M., Hradecky, P. and Wong, K.K., 1999. Targeted mutagenesis by duplication insertion in the radioresistant bacterium Deinococcus radiodurans: radiation sensitivities of catalase (katA) and superoxide dismutase (sodA) mutants. Journal of bacteriology, 181(2), pp.666669. 116. Marsh, V.L., Peak-Chew, S.Y. and Bell, S.D., 2005. Sir2 and the acetyltransferase, Pat, regulate the archaeal chromatin protein, Alba. Journal of Biological Chemistry, 280(22), pp.21122-21128. 117. Mazin, A.V., Mazina, O.M., Bugreev, D.V. and Rossi, M.J., 2010. Rad54, the motor of homologous recombination. DNA repair, 9(3), pp.286-302. 118. Middleton, E.M. and Teramura, A.H., 1993. The role of flavonol glycosides and carotenoids in protecting soybean from ultraviolet-B damage. Plant Physiology, 103(3), pp.741-752. 119. Mimitou, E.P. and Symington, L.S., 2009. DNA end resection: many nucleases make light work. DNA repair, 8(9), pp.983-995. 120. Minton, K.W., 1994. DNA repair in the extremely radioresistant bacterium Deinococcus radiodurans. Molecular microbiology, 13(1), pp.9-15. 121. Misra, C.S., Appukuttan, D., Kantamreddi, V.S.S., Rao, A.S. and Apte, S.K., 2012. Recombinant D. radiodurans cells for bioremediation of heavy metals from acidic/neutral aqueous wastes. Bioengineered, 3(1), pp.44-48. 122. Misra, H.S., Khairnar, N.P., Barik, A., Indira Priyadarsini, K., Mohan, H. and Apte, S.K., 2004. Pyrroloquinoline quinone: a reactive oxygen species scavenger in bacteria. FEBS letters, 578(1-2), pp.26-30.

123. Miyake, C., Michihata, F. and Asada, K., 1991. Scavenging of hydrogen peroxide in prokaryotic and eukaryotic algae: acquisition of ascorbate peroxidase during the evolution of cyanobacteria. Plant and Cell

Physiology, 32(1), pp.33-43. 124. Molina-Heredia, F.P., Houée-Levin, C., Berthomieu, C., Touati, D., Tremey, E., Favaudon, V., Adam, V. and Nivière, V., 2006. Detoxification of superoxide without production of H2O2: antioxidant activity of superoxide reductase complexed

Page 124

with ferrocyanide. Proceedings of the National Academy of Sciences, 103(40), pp.14750-14755. 125. Montero, O. and Lubián, L.M., 2003. Mycosporine-like amino acid (MAAs) production by Heterocapasa sp.(Dinophyceae) in indoor cultures. Biomolecular engineering, 20(4), pp.183-189. 126. Mori, H., Nishinaka, Y., Nonogawa, M., Sommani, P., Makino, K., Yamashita, K. and Arai, T., 2010. Substituent effects of pterin derivatives on singlet oxygen scavenging activity. Biological and Pharmaceutical Bulletin, 33(5), pp.905-908. 127. Mukherjee, P., Fulton, D.B., Halder, M., Han, X., Armstrong, D.W., Petrich, J.W. and Lobban, C.S., 2006. Maristentorin, a Novel Pigment from the Positively Phototactic Marine Ciliate Maristentor d inoferus, Is Structurally Related to Hypericin and Stentorin. The Journal of Physical Chemistry B, 110(12), pp.6359-6364. 128. Müller, K., 2001. Pharmaceutically relevant metabolites from lichens. Applied Microbiology and Biotechnology, 56(1-2), pp.9-16. 129. Murakami, M., Narumi, I., Satoh, K., Furukawa, A. and Hayata, I., 2006. Analysis of interaction between DNA and Deinococcus radiodurans PprA protein by atomic force microscopy. Biochimica et Biophysica Acta (BBA)Proteins and Proteomics, 1764(1), pp.20-23. 130. Nakayama, H., Yoshida, K., Ono, H., Murooka, Y. and Shinmyo, A., 2000. Ectoine, the compatible solute of Halomonas elongata, confers hyperosmotic tolerance in cultured tobacco cells. Plant physiology, 122(4), pp.1239-1248. 131. Narumi, I., Satoh, K., Cui, S., Funayama, T., Kitayama, S. and Watanabe, H., 2004. PprA: a novel protein from Deinococcus radiodurans that stimulates DNA ligation. Molecular microbiology, 54(1), pp.278-285. 132. Nguyen, H.H., La Tour, D., Bouthier, C., Toueille, M., Vannier, F., Sommer, S. and Servant, P., 2009. The essential histone like protein HU plays a major role in Deinococcus radiodurans nucleoid compaction. Molecular microbiology, 73(2), pp.240-252. 133. O'Brien, P.A. and Houghton, J.A., 1982. Photoreactivation and excision repair of UV induced pyrimidine dimers in the unicellular cyanobacterium Gloeocapsa alpicola (Synechocystis PCC 6308). Photochemistry and photobiology, 35(3), pp.359- 364.

Page 125

134. Omelchenko, M.V., Wolf, Y.I., Gaidamakova, E.K., Matrosova, V.Y., Vasilenko, A., Zhai, M., Daly, M.J., Koonin, E.V. and Makarova, K.S., 2005. Comparative genomics of Thermus thermophilus and Deinococcus radiodurans: divergent routes of adaptation to thermophily and radiation resistance. BMC Evolutionary Biology, 5(1), p.57. 135. Ordoñez, O.F., Flores, M.R., Dib, J.R., Paz, A. and Farías, M.E., 2009. Extremophile culture collection from Andean lakes: extreme pristine environments that host a wide diversity of microorganisms with tolerance to UV radiation. Microbial Ecology, 58(3), pp.461-473. 136. Osburne, M.S., Holmbeck, B.M., Frias Lopez, J., Steen, R., Huang, K., Kelly, L., Coe, A., Waraska, K., Gagne, A. and Chisholm, S.W., 2010. UV hyper resistance in Prochlorococcus MED4 results from a single base pair deletion just upstream of an operon encoding nudix hydrolase and photolyase. Environmental microbiology, 12(7), pp.1978-1988. 137. Owttrim, G.W. and Coleman, J.R., 1987. Molecular cloning of a recA-like gene from the cyanobacterium Anabaena variabilis. Journal of bacteriology, 169(5), pp.1824-1829. 138. Oyamada, C., Kaneniwa, M., Ebitani, K., Murata, M. and Ishihara, K., 2008. Mycosporine-like amino acids extracted from scallop (Patinopecten yessoensis) ovaries: UV protection and growth stimulation activities on human cells. Marine Biotechnology, 10(2), pp.141-150. 139. Paerl, H.W., 1984. Cyanobacterial carotenoids: their roles in maintaining optimal photosynthetic production among aquatic bloom forming genera. Oecologia, 61(2), pp.143-149. 140. Pang, Q. and Hays, J.B., 1991. UV-B-inducible and temperature-sensitive photoreactivation of cyclobutane pyrimidine dimers in Arabidopsis thaliana. Plant Physiology, 95(2), pp.536-543. 141. Pavlov, N.A., Cherny, D.I., Nazimov, I.V., Slesarev, A.I. and Subramaniam, V., 2002. Identification, cloning and characterization of a new DNA-binding protein from the hyperthermophilic kandleri. Nucleic acids research, 30(3), pp.685-694.

Page 126

142. Proteau, P.J., Gerwick, W.H., Garcia-Pichel, F. and Castenholz, R., 1993. The structure of scytonemin, an ultraviolet sunscreen pigment from the sheaths of cyanobacteria. Experientia, 49(9), pp.825-829. 143. Quaiser, A., Constantinesco, F., White, M.F., Forterre, P. and Elie, C., 2008. The Mre11 protein interacts with both Rad50 and the HerA bipolar helicase and is recruited to DNA following gamma irradiation in the archaeon Sulfolobus acidocaldarius. BMC molecular biology, 9(1), p.25. 144. Quesada, A. and Vincent, W.F., 1997. Strategies of adaptation by Antarctic cyanobacteria to ultraviolet radiation. European Journal of Phycology, 32(4), pp.335- 342. 145. Rainey, F.A., Ray, K., Ferreira, M., Gatz, B.Z., Nobre, M.F., Bagaley, D., Rash, B.A., Park, M.J., Earl, A.M., Shank, N.C. and Small, A.M., 2005. Extensive diversity of ionizing-radiation-resistant bacteria recovered from Sonoran Desert soil and description of nine new species of the genus Deinococcus obtained from a single soil sample. Applied and Environmental Microbiology, 71(9), pp.5225-5235. 146. Rao, M.V., Paliyath, G. and Ormrod, D.P., 1996. Ultraviolet-B-and ozoneinduced biochemical changes in antioxidant enzymes of Arabidopsis thaliana. Plant physiology, 110(1), pp.125-136. 147. Rastogi, R.P., Sinha, R.P., Singh, S.P. and Häder, D.P., 2010. Photoprotective compounds from marine organisms. Journal of industrial microbiology & biotechnology, 37(6), pp.537-558. 148. Russo, A., Piovano, M., Lombardo, L., Garbarino, J. and Cardile, V., 2008. Lichen metabolites prevent UV light and nitric oxide-mediated plasmid DNA damage and induce apoptosis in human melanoma cells. Life sciences, 83(13), pp.468-474. 149. Sakai, A. and Cox, M.M., 2009. RecFOR and RecOR as distinct RecA loading pathways. Journal of Biological Chemistry, 284(5), pp.3264-3272. 150. Sandman, K. and Reeve, J.N., 2006. Archaeal histones and the origin of the histone fold. Current opinion in microbiology, 9(5), pp.520-525. 151. Satyanarayana, T., Raghukumar, C. and Shivaji, S., 2005. Extremophilic microbes: diversity and perspectives. Indian Academy of Sciences. 152. Scherer, S., Chen, T.W. and Böger, P., 1988. A new UV-A/B protecting pigment in the terrestrial cyanobacterium Nostoc commune. Plant Physiology, 88(4), pp.1055- 1057.

Page 127

153. Seib, K.L., Tseng, H.J., McEwan, A.G., Apicella, M.A. and Jennings, M.P., 2004. Defenses against oxidative stress in Neisseria gonorrhoeae and Neisseria meningitidis: distinctive systems for different lifestyles. Journal of Infectious Diseases, 190(1), pp.136-147. 154. Seitz, E.M., Brockman, J.P., Sandler, S.J., Clark, A.J. and Kowalczykowski, S.C., 1998. RadA protein is an archaeal RecA protein homolog that catalyzes DNA strand exchange. Genes & development, 12(9), pp.1248-1253. 155. Servant, P., Jolivet, E., Bentchikou, E., Mennecier, S., Bailone, A. and Sommer, S., 2007. The ClpPX protease is required for radioresistance and regulates cell division after γ irradiation in Deinococcus radiodurans. Molecular microbiology, 66(5), pp.1231-1239. 156. Seyrig, G., 2010. Uranium bioremediation: current knowledge and trends. Basic Biotechnol eJournal, 6(1), p.3. 157. Shahmohammadi, H.R., Asgarani, E., Terato, H., Saito, T., Ohyama, Y., Gekko, K., Yamamoto, O. and Ide, H., 1998. Protective roles of bacterioruberin and intracellular KCl in the resistance of Halobacterium salinarium against DNA- damaging agents. Journal of radiation research, 39(4), pp.251-262. 158. Shashidhar, R. and Bandekar, J.R., 2006. Deinococcus mumbaiensis sp. nov., a radiation-resistant pleomorphic bacterium isolated from Mumbai, India. FEMS microbiology letters, 254(2), pp.275-280. 159. Shibata, H., Noda, T., Ogura, Y., Suginaka, K., Matsui, Y., Ozoe, Y., Sawa, Y. and Kono, Y., 1996. A soluble-form of pro-oxidant lumazine isolated from cyanobacterial cells generates superoxide anion under near-UV irradiation. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 1274(3), pp.129-134. 160. Shivaji, S., Reddy, G.S.N., Raghavan, P.U.M., Sarita, N.B. and Delille, D., 2004. Psychrobacter salsus sp. nov. and Psychrobacter adeliensis sp. nov. isolated from fast ice from Adelie Land, Antarctica. Systematic and applied microbiology, 27(6), pp.628- 635. 161. Shuman, S. and Glickman, M.S., 2007. Bacterial DNA repair by nonhomologous end joining. Nature Reviews Microbiology, 5(11), pp.852-861. 162. Singh, O.V. and Gabani, P., 2011. Extremophiles: radiation resistance microbial reserves and therapeutic implications. Journal of applied microbiology, 110(4), pp.851-861.

Page 128

163. Singh, O.V., 2006. Proteomics and metabolomics: The molecular make up of toxic aromatic pollutant bioremediation. Proteomics, 6(20), pp.5481-5492. 164. Singh, S.P., Klisch, M., Sinha, R.P. and Häder, D.P., 2010. Genome mining of mycosporine-like amino acid (MAA) synthesizing and non-synthesizing cyanobacteria: a bioinformatics study. Genomics, 95(2), pp.120-128. 165. Singh, S.P., Sinha, R.P., Klisch, M. and Häder, D.P., 2008. Mycosporine-like amino acids (MAAs) profile of a rice-field cyanobacterium Anabaena doliolum as influenced by PAR and UVR. Planta, 229(1), pp.225-233. 166. Slade, D. and Radman, M., 2011. Oxidative stress resistance in Deinococcus radiodurans. Microbiology and Molecular Biology Reviews, 75(1), pp.133-191. 167. Slade, D., Lindner, A.B., Paul, G. and Radman, M., 2009. Recombination and replication in DNA repair of heavily irradiated Deinococcus radiodurans. Cell, 136(6), pp.1044-1055. 168. Sorrels, C.M., Proteau, P.J. and Gerwick, W.H., 2009. Organization, evolution, and expression analysis of the biosynthetic gene cluster for scytonemin, a cyanobacterial UV-absorbing pigment. Applied and environmental microbiology, 75(14), pp.4861-4869. 169. Soule, T., Garcia-Pichel, F. and Stout, V., 2009. Gene expression patterns associated with the biosynthesis of the sunscreen scytonemin in Nostoc punctiforme ATCC 29133 in response to UVA radiation. Journal of bacteriology, 191(14), pp.4639- 4646. 170. Soule, T., Stout, V., Swingley, W.D., Meeks, J.C. and Garcia-Pichel, F., 2007. Molecular genetics and genomic analysis of scytonemin biosynthesis in Nostoc punctiforme ATCC 29133. Journal of bacteriology, 189(12), pp.44654472. 171. Steiger, S., Astier, C. and Sandmann, G., 2000. Substrate specificity of the expressed carotenoid 3, 4-desaturase from Rubrivivax gelatinosus reveals the detailed reaction sequence to spheroidene and spirilloxanthin. Biochemical Journal, 349(2), pp.635-640. 172. Stephen, J.R. and Macnaughtont, S.J., 1999. Developments in terrestrial bacterial remediation of metals. Current opinion in biotechnology, 10(3), pp.230-233. 173. Stevenson, C.S., Capper, E.A., Roshak, A.K., Marquez, B., Eichman, C., Jackson, J.R., Mattern, M., Gerwick, W.H., Jacobs, R.S. and Marshall, L.A., 2002. The identification and characterization of the marine natural product scytonemin as a

Page 129

novel antiproliferative pharmacophore. Journal of Pharmacology and Experimental Therapeutics, 303(2), pp.858-866. 174. Stevenson, C.S., Capper, E.A., Roshak, A.K., Marquez, B., Grace, K., Gerwick, W.H., Jacobs, R.S. and Marshall, L.A., 2002. Scytonemin-a marine natural product inhibitor of kinases key in hyperproliferative inflammatory diseases. Inflammation Research, 51(2), pp.112-114. 175. Summers, A.O., 1986. Organization, expression, and evolution of genes for mercury resistance. Annual Reviews in Microbiology, 40(1), pp.607-634. 176. Sun, Z., Shen, S., Tian, B., Wang, H., Xu, Z., Wang, L. and Hua, Y., 2009. Functional analysis of γ-carotene ketolase involved in the carotenoid biosynthesis of Deinococcus radiodurans. FEMS microbiology letters, 301(1), pp.21-27.

177. Sun, Z., Shen, S., Wang, C., Wang, H., Hu, Y., Jiao, J., Ma, T., Tian, B. and Hua, Y., 2009. A novel carotenoid 1, 2-hydratase (CruF) from two species of the non- photosynthetic bacterium Deinococcus. Microbiology, 155(8), p.2775. 178. Suresh, K., Reddy, G.S.N., Sengupta, S. and Shivaji, S., 2004. Deinococcus indicus sp. nov., an arsenic-resistant bacterium from an aquifer in West Bengal, India. International journal of systematic and evolutionary microbiology, 54(2), pp.457-461. 179. Takaichi, S. and Mochimaru, M., 2007. Carotenoids and carotenogenesis in cyanobacteria: unique ketocarotenoids and carotenoid glycosides. Cellular and molecular life sciences, 64(19-20), p.2607. 180. Tanaka, M., Earl, A.M., Howell, H.A., Park, M.J., Eisen, J.A., Peterson, S.N. and Battista, J.R., 2004. Analysis of Deinococcus radiodurans's transcriptional response to ionizing radiation and desiccation reveals novel proteins that contribute to extreme radioresistance. Genetics, 168(1), pp.21-33. 181. Tao, L. and Cheng, Q., 2004. Novel β-carotene ketolases from nonphotosynthetic bacteria for canthaxanthin synthesis. Molecular Genetics and Genomics, 272(5), pp.530-537. 182. Tao, L., Picataggio, S., Rouviere, P.E. and Cheng, Q., 2004. Asymmetrically acting lycopene β-cyclases (CrtLm) from non-photosynthetic bacteria. Molecular Genetics and Genomics, 271(2), pp.180-188. 183. Tapias, A., Leplat, C. and Confalonieri, F., 2009. Recovery of ionizingradiation damage after high doses of gamma ray in the hyperthermophilic archaeon Thermococcus gammatolerans. Extremophiles, 13(2), pp.333-343.

Page 130

184. Tapias, A., Leplat, C. and Confalonieri, F., 2009. Recovery of ionizingradiation damage after high doses of gamma ray in the hyperthermophilic archaeon Thermococcus gammatolerans. Extremophiles, 13(2), pp.333-343. 185. Tease, B.E. and Walker, R.W., 1987. Comparative composition of the sheath of the cyanobacterium Gloeothece ATCC 27152 cultured with and without combined nitrogen. Microbiology, 133(12), pp.3331-3339. 186. Terato, H., Suzuki, K., Nishioka, N., Okamoto, A., Shimazaki-Tokuyama, Y., Inoue, Y. and Saito, T., 2011. Characterization and radio-resistant function of manganese superoxide dismutase of Rubrobacter radiotolerans. Journal of radiation research, 52(6), pp.735-742. 187. Thomas, D.N. and Dieckmann, G.S., 2002. Antarctic sea ice--a habitat for extremophiles. Science, 295(5555), pp.641-644. 188. Tian, B. and Hua, Y., 2010. Carotenoid biosynthesis in extremophilic Deinococcus– Thermus bacteria. Trends in microbiology, 18(11), pp.512-520. 189. Tian, B., Sun, Z., Xu, Z., Shen, S., Wang, H. and Hua, Y., 2008. Carotenoid 3′, 4′- desaturase is involved in carotenoid biosynthesis in the radioresistant bacterium Deinococcus radiodurans. Microbiology, 154(12), pp.3697-3706. 190. Tian, B., Xu, Z., Sun, Z., Lin, J. and Hua, Y., 2007. Evaluation of the antioxidant effects of carotenoids from Deinococcus radiodurans through targeted mutagenesis, chemiluminescence, and DNA damage analyses. Biochimica et Biophysica Acta (BBA)-General Subjects, 1770(6), pp.902-911. 191. Torres, A., Enk, C.D., Hochberg, M. and Srebnik, M., 2006. Porphyra-334, a potential natural source for UVA protective sunscreens. Photochemical & Photobiological Sciences, 5(4), pp.432-435. 192. Vaishampayan, P., Probst, A., Krishnamurthi, S., Ghosh, S., Osman, S., McDowall, A., Ruckmani, A., Mayilraj, S. and Venkateswaran, K., 2010. Bacillus horneckiae sp. nov., isolated from a spacecraft-assembly clean room. International journal of systematic and evolutionary microbiology, 60(5), pp.1031-1037. 193. van Hullebusch, E.D., Peerbolte, A., Zandvoort, M.H. and Lens, P.N., 2005. Sorption of cobalt and nickel on anaerobic granular sludges: isotherms and sequential extraction. Chemosphere, 58(4), pp.493-505.

Page 131

194. Vertuani, S., Angusti, A. and Manfredini, S., 2004. The antioxidants and proantioxidants network: an overview. Current pharmaceutical design, 10(14), pp.1677-1694. 195. Walker, G.C., 1985. Inducible DNA repair systems. Annual review of biochemistry, 54(1), pp.425-457. 196. Wang, W., Mao, J., Zhang, Z., Tang, Q., Xie, Y., Zhu, J., Zhang, L., Liu, Z., Shi, Y. and Goodfellow, M., 2010. Deinococcus wulumuqiensis sp. nov., and Deinococcus xibeiensis sp. nov., isolated from radiation-polluted soil. International journal of systematic and evolutionary microbiology, 60(9), pp.2006-2010. 197. Weller, G.R., Kysela, B., Roy, R., Tonkin, L.M., Scanlan, E., Della, M., Devine, S.K., Day, J.P., Wilkinson, A., di Fagagna, F.D.A. and Devine, K.M., 2002. Identification of a DNA nonhomologous end-joining complex in bacteria. Science, 297(5587), pp.1686-1689. 198. White, M.F. and Bell, S.D., 2002. Holding it together: chromatin in the Archaea. TRENDS in Genetics, 18(12), pp.621-626. 199. Whitehead, K. and Hedges, J.I., 2005. Photodegradation and photosensitization of mycosporine-like amino acids. Journal of Photochemistry and Photobiology B: Biology, 80(2), pp.115-121. 200. Williams, E., Lowe, T.M., Savas, J. and DiRuggiero, J., 2007. Microarray analysis of the hyperthermophilic archaeon Pyrococcus furiosus exposed to gamma irradiation. Extremophiles, 11(1), pp.19-29. 201. Wu, W.M., Carley, J., Gentry, T., Ginder-Vogel, M.A., Fienen, M., Mehlhorn, T., Yan, H., Caroll, S., Pace, M.N., Nyman, J. and Luo, J., 2006. Pilot-scale in situ bioremedation of uranium in a highly contaminated aquifer. 2. Reduction of U (VI) and geochemical control of U (VI) bioavailability. Environmental science & technology, 40(12), pp.3986-3995.

202. Wyman, C. and Kanaar, R., 2006. DNA double-strand break repair: all's well that ends well. Annu. Rev. Genet., 40, pp.363-383. 203. Yeeles, J.T. and Dillingham, M.S., 2010. The processing of double-stranded DNA breaks for recombinational repair by helicase–nuclease complexes. DNA repair, 9(3), pp.276-285. 204. Yun, N.R. and Lee, Y.N., 2009. Iso-superoxide dismutase in Deinococcus grandis, a UV resistant bacterium. The Journal of Microbiology, 47(2), pp.172177.

Page 132

205. Zahradka, K., Slade, D., Bailone, A., Sommer, S., Averbeck, D., Petranovic, M., Lindner, A.B. and Radman, M., 2006. Reassembly of shattered chromosomes in Deinococcus radiodurans. Nature, 443(7111), pp.569-573. 206. Zhang, L., Yang, Q., Luo, X., Fang, C., Zhang, Q. and Tang, Y., 2007. Knockout of crtB or crtI gene blocks the carotenoid biosynthetic pathway in Deinococcus radiodurans R1 and influences its resistance to oxidative DNAdamaging agents due to change of free radicals scavenging ability. Archives of microbiology, 188(4), pp.411-419. 207. Zimmerman, J.M. and Battista, J.R., 2005. A ring-like nucleoid is not necessary for radioresistance in the Deinococcaceae. BMC microbiology, 5(1), p.17.

Page 133

Chapter 3: Isolation and Characterization of Radio-resistant Bacteria

Paper 1

Title: Isolation and Characterization of Ultra Violet Rays (UVR) Resistant Bacteria from Desert Soil Samples of Pakistan

Page 134

3.1. Abstract

Living beings or dwellers of xeric environments are able to withstand adverse life circumstances, desiccation, and extreme temperatures, sturdy thermal gaps and nutrient deficit. The phylogenetic diversity of ultra-violet (UV) resistant bacteria from desert soil, was investigated by culture and molecular based analysis. The bacterial strains were observed for their tolerance to UV doses, salt concentration, and heavy metals. The effect of UV radiation on cellular protein and lipids was also investigated. A total of 09 UV resistant bacteria were isolated and identified through biochemical tests and 16S rRNA sequencing. Based on the results obtained, bacterial strains were assigned to four phyla: Firmicutes, Proteobacteria, Deinococcus-Thermus and Actinobacteria. High UV survivability was observed in case of genus Deinococcus followed by Firmicutes. The bacteria were found to grow at wide temperature and pH range, resistant to high salt concentration as well as various metal ions. The microbes exhibited minor damages to protein and lipids as a result of exposure to UV radiation as compared to Escherichia coli (ATCC 10536). The results indicated that these microbes harbor a sophisticated phenotypic character and molecular repair mechanisms that can prolong their survival in extreme radiations.

Key words: Radio-resistant, Phylogenetic analysis, Deinococcus-Thermus, Protein carbonylation, Lipid peroxidation

3.2. Introduction

Radio-resistant is the term referred to the group of organisms that live under radiation conditions. These organisms surprisingly can endure both ionizing and nonionizing radiations, which could be lethal to others (Singh and Gabani, 2011). Diverse environments which encompass dried food, irradiated meat and fish, high level nuclear wastes at Savannah River in South Carolina, hot and dry desert, and warm fresh water, geothermal spring and at Hanford in Washington have been investigated for isolation of ionizing-radiation resistant microbes. Numerious members of the domains Archaea and Bacteria have demonstrated extreme ionizing radiation resistance (Fredrickson et al., 2004) which appears to be an attribute of the organisms belonging to genera, Deinococcus and Rubrobacter followed by Kineococcus, and Kocuria (Phillips et al., 2002).

Bacteria in the extreme Ultraviolet radiation (UVR) environment is consider on of the crucial exogenous stress factor The interest of several environmental photobiology studies is concerned with the effects of UVB on bacteria. Exposure to solar UVR is responsible for ROS induced oxidative stress in aquatic bacteria synthesized via photo-dynamic reactions which are continuously involved in intracellular or extracellular photo-sensitization (Pattison et al., 2006). The most important cellular target for ROS is proteins and lipids. These attacks the polyunsaturated fatty acids in cell membranes and initiate lipid peroxidation, which is accompanied by a decrease in membrane permeability and interference of transmembrane

Page 135

ion gradients that eventually leads to cell death. The deleterious effects of ROS are significantly influenced by metal ion homeostasis (Halliwell et al., 2015). In bacterial cells which are exposed to UV-B radiations, the potential synergistic effect of some transition metals like Cu+2, Mn+2 and Zn+2 are also been reported (Santos et al., 2013a). Intercellular Cu+2 acquisition, presence of high intracellular Mn+2 and Zn+2 uptake by UV resistant microbes are the adaptive response to peroxides stress by blocking the Fenton and Haber– Weiss reactions (Bagwell et al., 2008; Daly et al., 2010). Production of different compatible solutes trehalose and ectoine in high salt concentration play a significant role in ionization- radiation protection. Ionizingradiation resistance in Halobacterium salinarum, demonstrating that “metabolic route” with a combination of tightly coordinated physiological processes contributes to irradiation resistance (Robinson et al., 2011). These organisms also tend to produce metabolites during their defense against radiations. Recent biotechnological development helps to identify microbial approach of self-engineering to tolerate extreme ultra-violet radiations. Therefore, in order to explore the biological mechanisms involved in the survival under UV radiations, it is necessary to indicate the diversity of ultra-violet resistant microbes.

The current study focuses on determination of phylogenetic diversity of UV resistant bacteria isolated from sand samples of Lakki Marwat and Bahawalpur deserts, Pakistan. The effect of UVB radiations on the intracellular lipids and proteins of UV resistant bacterial isolates was also investigated by use of standard oxidation assays.

3.3. Materials and Methods

3.3.1. Sampling

Strictly adhering to microbiological standard procedures, soil samples to a depth of 15cm were accumulated aseptically from Lakki Marwat and Bahawalpur deserts, Pakistan, placed in sterilized polyethylene zipper bags and were carefully taken back to the lab of Department of Microbiology, Quaid-I-Azam University, Islamabad, and stored at 4°C for further processing.

3.3.2. Metal Analysis of Desert Soil

3.3.2.1. Sample Preparation

The soil samples were dried at room temperature for 5 days and sieved (2 mm sieve). 1g of the soil was acidified with 05 ml of concentrated Nitric acid and 10 ml of per- chloric acid (70% HClO4). The mixture was heated till white, dense fumes of HClO4 appeared

(RAURET 1998). The digested samples were cooled to room temperature, filtered through

Page 136

Whatman # 41 and boiled to remove oxides of nitrogen and chlorine. Finally, the soil samples were subjected to Cu+2, Ni+2, Zn+2, Mn+2, Cr+2,

Fe+2, Pb+2, Cd+2, Ca+2, Mg+2 and Na+2 analysis by Atomic Absorption Spectrophotometer (AAS) on a Perkin-Elmer 460 Spectrophotometer.

3.3.3. Isolation of Radio-resistant Microorganisms

The soil samples were serially diluted and plated on TGY (tryptone glucose yeast extract) agar by spread plate method. The plates were exposed to UV radiation in 119x69×52 cm UV chamber supplied with 20W and 280nm UV light source

(germicidal lamp) for a specific time (30-300 seconds). The UV fluence rate

(energy/area/time) to the test sample was measured with He=Ee×t in units of J/m2 (SAJJAD et al. 2017). The total UV dose was determined by time of exposure UV fluence rate. All UV irradiation procedures were performed under red light to prevent photo-reactivation.

Radiant exposure (He) = the energy that reaches a surface area due to irradiance (Ee) maintained for a time duration (t).

3.3.3.2. Radiant exposure calculation

Exposure of each TGY plates to UV radiation for 5 min was accomplished in a UV chamber (119x55×52 cm) prior to incubation, which conferred a 20W, 280 nm UV light placed at the top.

=0.52 mm

= 0.55 mm

Page 137

I=Ee

Now radiant exposure He=Ee×t

That is the radiant exposure (He) which can be define as irradiance (Ee) of the plate in time (t)

Where watt

If the exposure of the respective sample on the plate is 30 sec then,

So the Radiant exposure

3.3.4. UV Radiation Tolerance

The UVR resistance among bacterial isolates was determined by the method as described by Mattimore and Battista, (1996) with some modifications in order to find out

Page 138

survival curve. The UV-resistant bacteria isolated during preliminary screening were grown in

TGY broth to an OD600 0.5, and then spread on TGY agar. The plates were exposed to UV-B (280 nm) for the variable doses (30-180 sec) and subsequently incubated at 37ºC. The surviving fraction was calculated after 24hrs by determining the titer of culture after irradiation divided by un-irradiated control.

3.3.5. Identification of UV Resistant Microbes

3.3.5. Biochemical and Physiological Characteristics

Phase contrast microscopy was utilized to determine the cellular morphology of living cells (usually contained in culture) (Labomed Lx400). The bacteria were grown on TGY agar plates at wide temperature (15-45°C), pH (4.0-9.0) and NaCl (016%) for 3 days to determine optimum growth conditions. Moreover, the strains were also assessed for different biochemical tests such as catalase, cytochrome oxidase as well as starch hydrolysis, casein, and gelatin according to the procedures outlined by Murray et al., (1981).

3.3.5.2. Sequence Alignment and Phylogenetic Analysis All bacterial strains were subjected to basic steps of genomic DNA extraction facilitated by DNA extraction kit (QIAGEN). Following amplification of conserved

16S rRNA gene sequences, using universal primer (F27:AGAGTTTGATCMTGGCTCAG, R- 1492:TACGGYTACCTTGTTACGACTT) via PCR, amplicons were sequenced at Macrogen Service Center (Geunchun-gu, Seoul, South Korea). The obtained sequences were computed for closest relatives using BLAST tool at the NCBI database and homologs were analyzed for phylogeny using Molecular Evolutionary Genetic Analysis (MEGA) version 6 (Tamura et al.,

2013). A neighbor-joining tree was constructed based upon distance matrix, for identification and diversity among UVR resistant extremophiles with naturally occurring microorganisms.

3.3.6. Metal Resistance

Stock solutions (1000ppm) of various transition metals (Co+2, Cu+2, Fe+2, Mn+2, Cd+2, Hg+2, Ar+2, Cr+2 and Zn+2) were prepared in deionized, filter-sterilized water from the corresponding metallic salts. Effect of metal ions on bacterial strains was determined by inoculating them on TGY agar supplemented with different metals at variable concentrations (20-400 ppm) and incubated at 30°C for 48hrs.

3.3.7. Effect of UVB on Proteins and Lipids of UV Resistant Bacteria

Overnight grown culture in TGY broth was harvested by centrifugation at 10,000 rmp for 10 mins. The pallets of respective bacterial strain (106 cells/ml) was irradiated with UVB.

Page 139

The UV dose (2000 J/m2) was calculated by method described previously (Sajjad et al. 2017). An aliquot of cell suspension was collected before and after irradiation, washed with ultrapure water, and immediately used for lipid and protein extraction.

3.3.7.1. Lipid Extraction The entire steps of total lipid extraction were performed according to standard protocols (Baligh et al., 1959). Lipid extracts from irradiated and un-irradiated bacterial cells were centrifuged (13000×g for 10min, 4°C) and collected and washed with with pure sterile desalted water. The mixture was vortexed for 5 minutes and dissolved in chloroform (125 μL), methanol (250 μL). This step was repeated 3 times to ensure the extraction procedure. Finally 6 M HCl (8.4 μL) and chloroform (125 μL) was added and the mixture containing cell pellets was vortexed adding pure desalted water (125 μL). Upon centrifugation (20 min at 3000×g, 4°C) the total lipid extract was collected in the lower phase and aimed to prompt quantification by Malondialdehyde assay (MDA). All reagents used for analysis were procured from Sigma (St. Louis, MO).

3.3.7.2. Protein Extraction Cell suspensions comprised of both Irradiated and un-irradiated, were centrifuged and the pellets were redissolved in 10 Mm Tris-HCl (pH 8.0). the reaction mixture was sonicated on ice bath, four times for 5 s (Branson 450, Danbury, CT) and centrifuged again (15000×g, 10 minutes, at 4 °C). Finally, adding sarcosyl (1.5%, v/v) and 10 mM Tris-HCl (pH 8.0). The mixture was incubated at room temperature (20 min), and collecting the protein extract by centrifugation (15000×g, 90 min, 4°C). The cell proteins extracted were suspended in ultrapure water for quantification of degraded products facilitated by 2,4- Dinitrophenylhydrazine (DNPH) assay. All reagents were procured from Sigma (St. Louis, MO).

3.3.8. Lipid Peroxidation Assay

Lipid peroxidation results in formation of malondialdehyde (MDA), a lipid peroxidation marker. The TBA (Thiobarbituric acid) assay was performed by the method elucidated by Perez et al., (2007) with some modifications, to assess the MDA concentration.Following standard guidelines encouraged the total lipid extraction

(Bligh et al., 1959). 250 μL of lipid samples from irradiated and un-irradiated cultures was mixed with 125μL of 20% trichloro-acetic acid. Supernatant was collected, mixed with 0.5 mL

FeSO4 (0.07 M) and incubated at 37°C for 1hr. 300 μL of this solution was mixed with 0.8% TBA reagent (200 μL), 8% SDS (200 μL) and incubated at 100°C for 60min. The absorbance of chromophore developed was measured at 535 nm using UV sensitive E. coli (ATCC 10536) as a control. The MDA concentration is presented as mM of MDA produced per mg of lipids using a molar extinction coefficient of 1.56×105 M-1cm-1.

3.3.9. Intracellular Protein Carbonylation

Page 140

Generated ROS promoted protein oxidation i.e “protein carbonylation” which was detected by using a chemical compound DNPH (2,4-dinitrophenyl hydrazine) (Misra et al., , 2004). Centrifugation of both irradiated and un-irradiated cell suspensions were done and the pellets were re-dissolved in 10 mM Tris-HCl (pH 8.0), sarkosyl (1.5% v/v) and incubated at room temperature for 20 min. The total protein contents were quantified standard protocol procedure of Lowry et al., (1951). 2mg/ml of the protein extract was incubated with 50mM PBS (pH 7.4) and 400 µL of 10mM DNPH in 2 M HCl for 2 hrs in dark. The precipitated proteins in the reaction mixture was re-suspended in guanidine hydrochloride (6 M). The unbound proteins were washed with a mixture of ethyl acetate and ethanol 50%. The light transmittance of the supernatant was analyzed using spectrophotometer at a range of 370 nm. A protein control was run in parallel where DNPH was replaced with 2 M HCl. The protein carbonyl content was expressed in standard units “mM/mg” of protein.

3.3.10. Statistical Analysis

Appropriate statistical examination of results was evaluated by Student’s ttest,and P value or calculated probability <0.05 was considered as significant. Bacterial cell sensitivities were studied by plotting the data between the %survivability and their respective UV doses (Jm-2). Single factor and two-way ANOVA applied for analysis between and within groups.

3.4. Results.

3.4.1. Metal Analysis of Soil Samples

High solar radiation and large temperature oscillations between day and night were attributes of the sampling areas that were representatives of Lakki Marwat and Bahawalpur deserts. Figure 3.1 presents physiochemical analysis of soil samples harvested from two different deserts. Both the samples presented a higher concentration of Mn+2 followed by Mg+2, Fe+2 and Pb+2 while lower concentrations of Cd+2, Cu+2 and Cr+2 ion.

3.4.2. Isolation of Ultraviolet Rays (UVR) Resistant Microbes

Following exposure of the soil samples to different doses of UV radiation from 30 sec to 300 second with an energy dose ranges from 300-3300 J/m2. After incubation at 30°C for a week, nine representative colonies were selected, morphological characteristics were noted and purified on the same medium. Ultraviolet rays (UVR) resistant isolates were processed for UV, metal, salt and pH resistance and were also identified morphologically biochemically and molecular characterization based on 16S rRNA sequencing.

3.4.3. Resistance to UVB in Correlation with % Survivability

Selected isolates were analyzed for resistance to ionizing radiation. Radiotolerance among the isolates was ascertained by exposing each isolate to the UV-B radiation dosage at which they were isolated (300-3300 J/m2). Following prolonged period of UV-B exposure to

Page 141

soil samples, it was found that samples exposed to UVR possessed a significantly lower number of bacterial colony-forming units when compared to samples not exposed to UV-B radiation over the whole study period (P< 0.01) (Fig. 3.2). The initial dose of UV-B LD50 (2.0×103 J/m2) was found to be lethal for most of the isolates and determines the bulk of dose indispensable to kill 50 percent of a tested population. It was noted that with an increase of UV dose the CFU count decreases and these ultraviolet rays (UVR) resistant microbes can withstand to UV dose up to a certain extent, after that dose when gain there is a rapid decline observed in individual microbe. Out of all the 9 isolates WMA-LM19 was most sensitive and can withstand up to 1.30x103 J/m2energy. In case of WMA-LM9, WMA-LM30 and WMA-BD1 the percent survivability was noted as 79%, 68%, and 45%, even after receiving high doses of radiation and were considered as most potent UV resistant microbes.

3.4.4. Identification of UV Resistant Bacterial Strains

3.4.4.1. Morphology A highly diverse kind of bacteria was observed on un-irradiated TGY plates as indicated by their colony morphology, in comparison to the irradiated TGY plates usually comprised of colonies of different colors such as yellow, orange, pink, or red. There was Inverse relationship established between UV dose and CFU, demonstrated that Increase in doses of UV radiation led to the decrease in the number of CFU/g retrieved from the soil samples. After UV irradiation the UVR resistant isolates were examined morphologically and microscopically Table 1 shows the cellular morphology and Gram’s reaction of all the UV resistant bacteria from deserts sample. 3.4.4.2. Biochemical and Physiological Characteristics of UV Resistant Microbes Biochemical, physiological and other characteristics, i.e. temperature, pH range and salt tolerance of ultraviolet rays (UVR) resistant microbes were also investigated. Table 2 shows biochemical and other physiological characteristics of these isolates. Results revealed that all these isolates have a good potential to grow at high salt concentration ranges from 2% to 16 with a broad pH (6-10), temperature (20-45) range and capability of producing different hydrolytic enzymes like amylase, protease, gelatinase, DNase and others shown in the table 2. 79% of the UVR isolates showed activity of different hydrolytic enzymes.

3.4.4.3. Molecular Characterization and Phylogenetic Analysis of UVR Bacteria Partial sequencing of 16S rRNA gene of 9 isolates, representing dominant morphotype cultured on plates, led to the assessment of phylogenetic heterogenity. The tree with the highest log likelihood (-2463.3793) is shown. The analysis involved 54 nucleotide sequences; 462 positions while all missing data and gaps already eliminated. Evolutionary analysis was conducted in software MEGA 6.0. Assigned accession numbers of the associated sequences are presented in parentheses after the strain designation. Numbers at nodes are percentage bootstrap values based on 1,000 replications. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site (Fig. 3.3).

The sequences derived are associated to four bacterial phyla: Actinobacteria (1 isolates), Proteobacteria (3 isolates), Firmicutes (3 isolates) and DienococccusThermus group

Page 142

(2 isolates). Among these isolates, majority (8 out of 9) manifested close relationships (99% of similarity) with well-identified species that are widely disseminated in soil (see Table 1). Proeteobacteria are dominated by 3 species shown distant relationships to the genera Stenotrophomonas, (93–99% of similarity). Two isolates were clustered near phylum Deinococcus-Thermus and showed 99% similarity to Deinococcus sp. which is considered to be among the most UV resistant organisms up till now. While three Firmicutes isolates clustered near the genera Bacillus (2) and Staphylococcus (1) with 99% similarity (Fig. 3.3). Finally,

Actinobacteria comprised of only one isolate that showed minute relatedness to genus Kocoria (99% similarity), as shown in table 3. It is seeking attention that the 16S rRNA gene sequences of the 9 isolates and database sequences, obtained from different environments(arid, semi-arid environments and polluted soils) have close relatedness. (Table 2.1)

3.4.4.4. Nucleotide Sequence and Excision Number Pure culture -based driven 16S rRNA sequences were submitted in the GenBank database with following accession numbers: KT008382 (Stenotrophomonas maltophilia WMA-LM10); KT008383 (Stenotrophomonas sp. WMA-LM19);, KT008384 (Deinococcus sp. WMA-LM30);, KT008385, (Bacillus licheniformis

WMA-BD2); KT008386 (Staphyphylococcus lugdunensis WMA-BD4); KT008387 (Kocuria turfanensis WMA-BD1); KT008388 (Bacillus pumilus WMA-LM4); KT008389 (Deinococcus radiopugnans WMA-LM9); KT008390 (Bacillus subtilis WMA-LM15).

3.4.5. Minimum Inhibitory Concentration of UVR Microbes to Different Metals

Preliminary experiments were conducted with different metal concentrations. The majority of the UV resistant strains were sensitive to Hg+2, WMA-LM10 is considered to be more resistant to Hg+2 (40ppm). All isolates were more resistant to Mn+2, Co+2, Cr+2, and Ni+2, which can be directly correlated to UVB resistance. In the presence of Mn+2, an interesting change in cultural characteristics of strain WMALM9, WMA-LM30 and WMA-LM19 was observed, that is an increase in size of the colony and brighter coloration. Some strains, for instance, WMA-BD1, WMA-LM15, WMA-LM9, WMA-LM30 (240-280 ppm) had demonstrated greater potential for intracellular Cu ion sequestration that displayed a protective barrier against the detrimental effects of ionizing radiation. Mn+2 ions also safeguard against oxidative damage induced by several exogenous stresses, including UV-B, gamma-irradiation, wet and dry heat and H2O2.

3.4.6. Effect of UVB on Whole Cell Proteins

Page 143

Effect of UVB on the whole cell protein was measured, UV resistant isolates provide a strong protection to cellular protein as compared to E. coli (ATCC 10536) that is a UV sensitive strain run as a control. Strain WMA-LM9, WMA-LM30, WMA-BD1 and WMA-BD2 shown in the figure have a strong potential to survive in high UV dose and protected their protein. An increase in total intracellular protein was observed in strain WMA-LM4 (Fig. 5).

Page 144

Table 3.1: Microscopic characteristics with cultural morphology of UV resistant bacteria isolated from desert soil samples. Culture code Sampling site Morphology Microscopy

WMA-BD1 Bahawalpur Small to medium sized light pink colored mucoid G (+) cocci desert and circular, raised colonies with entire margins

WMA-BD2 Bahawalpur Large light off-white oval shaped colonies dry G (+) rods desert surface, forming crystal like structure when aggregates

WMA-BD4 Bahawalpur Medium to large yellow colored circular raised G (+) rods desert colonies with entire margins

WMA-LM4 Lakki Marwat Small to medium sized off white smooth and G (+) rods desert circular, flat colonies with entire margins

WMA-LM9 Lakki Marwat Medium brick red colored colonies mucoid circular G (+) cocci desert with entire margins

WMA-LM10 Lakki Marwat Large flat off white in color with dry surface G (˗) rods desert colonies with irregular margins

WMA-LM15 Lakki Marwat Large flat off white in color oval shape dry colonies G (˗) rods desert with entire margins

WMA-LM19 Lakki Marwat Large off white in color, flat colonies with shiny G (˗) rods desert surface, circular with entire margins

WMA-LM30 Lakki Marwat Medium brick red colored colonies with dry G (˗) cocci desert surface, circular with entire margins occur singly or tetrads

Page 145

Figure: 3.1: Metal analysis (in ppm) of soil samples collected from deserts. No significant difference was observed among the groups as p>0.05

Figure 3.2: Survivability of total UVR resistant isolates from desert soil at varying UV-B exposure. % survivability was measured using the formula N1/N0*100. N1 is the number of colonies after UV irradiation while N0 number of colonies after UV irradiation. Statistically it is proved that there is a significant effect of dose level (Jm2) on UVR isolates and significant effect of UV on various strains at different time exposure (p<0.05).

Page 146

Table 3.2: Biochemical and physiological characteristics of UV Resistant isolates from Lakki Marwat and Bahawalpur desert soil.

Characteristics Bacterial Strains

WMA- WMA- WMA- WMA- WMA- WMA- WMA- WMA- WMA- LM9 LM4 LM15 LM19 LM30 BD1 LM10 BD2 BD4

Temperature 10-30 25-37 25-45 20-45 10-30 20-35 20-45 25-40 20-37 pH 7-8 7-9 6-9 6-10 7-8 7-9 6-10 7-10 7-9

Salt tolerance 2% 14% 16% 12% 6% 12% 10% 10% 10%

Catalase + + + + + + + + +

Oxidase - - + + - - + - -

Amylase + - + + - + + + +

Protease + + + + + - + + +

Gelatinase + - + + + + + + -

DNase + - + + - + + - -

Table 3.3: 16S rRNA sequence homologues, closest related species, % survivability, gene bank accession number and query coverage of Ultraviolet radiation (UV subtype –B) resistant isolates from desert samples.

Page 147

Isolates GenBank Closest related Query similarity UVR Survival Score* % Accession Species Coverage resistance Rate % Number (%) J/m2

WMA-BD1 KT008387 Kocuria 100 99 3.3×103 45.45 turfanensis

WMA-BD2 KT008385 Bacillus 100 99 2.0×103 43.18 licheniformis

WMA-BD4 KT008386 Staphylococcus 100 99 2.0×103 48.27 lugdunensis

WMA-LM4 KT008388 Bacillus pumilus 99 99 2.60×103 45.28

WMA-LM9 KT008389 Deinococcus 100 99 3.30×103 79.47 radiopugnans

WMA-LM10 KT008382 Stenotrophomonas 100 99 1.30×103 46.15 maltophilia

WMA-LM15 KT008390 Bacillus subtilis 100 99 3.30×103 38.72

WMA-LM19 KT008383 Stenotrophomonas sp. 99 93 1.30×103 51.69

WMA-LM30 KT008384 Deinococcus sp. 100 100 3.30×103 68.03

Page 148

Figure 3.3: Phylogenetic analysis of UV resistant bacterial strains by maximum likelihood method based on Tamura-Nei model (1993). Bar=0.05 sequence variation.

Page 149

3.4.7. Protein and Lipid Oxidation of UVR Isolates

UVB can strongly affect the cell lipids and proteins and can cause a serious damage to cells. The effect of UV on cellular protein oxidation and lipid per-oxidation was measured using E. coli (10536) UV sensitive strain. E. coli (10536) displayed a significant damage to its cellular lipids and protein upon UV treatment with lipid oxidation of 12 µM and protein oxidation 189 mM/mg. Figure 2.5 revealed that strain WMA-LM9, WMA-LM30 and WMA- BD1 having a strong scavenging system to

detoxify the different superoxide’s that can damage the cell protein and lipids. A lower lipid oxidation (6.3µM) and protein oxidation (128mM/mg) was measured in WMA-LM9 followed by WMA-LM30 (8 µM, 138 mM/mg) and WMA-BD1 (10 µM, 135 mM/mg) respectively.

Figure 3.4: Effect of UVB on total cell protein content in mg/ml of radio-resistant bacteria from desert soil samples.

Table 3.4: Effect of the metal ions (in ppm) on growth of UVR resistant bacteria from desert samples on TGY agar plates. Values are shown in ppm.

Strain code Cd+2 Zn+2 Cr+2 Fe+2 Cu+2 Ni+2 Hg+2 Ar+2 Mn+2

Page 150

WMA-BD1 380 160 360 360 280 280 10 320 300

WMA-BD2 200 200 320 360 200 200 10 300 260

WMA-BD4 240 240 300 360 200 280 20 300 240

WMA-LM4 360 280 360 300 240 280 0 280 200

WMA-LM9 200 80 380 280 200 280 0 200 220

WMA-LM10 240 200 300 360 200 280 40 300 300

WMA-LM15 220 280 360 360 240 360 0 320 340

WMA-LM19 280 120 280 360 200 200 10 300 280

WMA-LM30 280 200 360 200 280 380 0 280 340

Page 151

Figure 3.5: Protein oxidation to quantify carbonylated protein and lipid peroxidation assay for TBARS in UV treated isolates from desert soil.

Page 152

3.5. Discussion

The soil samples were collected in June-July from Lakki Marwart and Bahawalpur deserts in Pakistan. Few environmental studies have been investigated by scientists on the bacterial community inhabiting the extreme environment like high hypersaline environment and high UV radiations, no screening programs have been designed to explore these UV resistant microbes for different hydrolytic enzymes and other therapeutic agents of biotechnological use. These microbes can not only be a potential source for enzyme industries, but also their secondary metabolic products by which these microbes prevent themselves from high UV dose could be a potential source of drugs for aging and cancer. The high correlation of UV, salt and metal's resistance investigated in this study will make the readers more attractive to investigate the molecular mechanisms of this synergistic effect in the microbes for UV resistant. The physiochemical analysis of the soil samples collected from these deserts indicated that Mn+2, Mg+2, Fe+2 and Pb+2 were the most abundant ion followed by Ca+2, Ni+2, Na+2 and Zn+2. Studies associated with scrutinization of several biological systems have validated the significant role exhibited by transition metal ions in protecting against the damaging effect of radiations, wet and dry heat and H2O2 (Daly et al., 2004; Bagwell et al., 2008; Ghosh et al., 2011). Desert varnishing contributes to the existence of high levels of Mn+2 in sand formation. During the process of sand formation in the desert, the occurrence of manganese oxide in rock varnish has an efficacious role in obstructing the transmission of ultraviolet radiation. Perhaps the microbes which dwell in the rock have attributes of synthesizing their own manganese-formula sunscreen. The bulk of varnish together with manganese oxides confers the dark color to desert soil (Fleisher et al., 1999).

When 16S rRNA gene sequences were compared, most of the UVB resistant bacteria were gram positive and assigned to four different clusters. 47% of the ultraviolet resistant isolates were from Fermicutes phylum followed by Deinocuccos the most UV resistant genera, Proteobacteria and Actinobacteria Deinococcus thermus and Actinobacteria showed 99% similarity to Deinococcus sp. and Kocuria turfanensis Gram positive pink colored bacteria on TGY agar. Our findings were accordance to (Flores et al., 2009; Moreno et al., 2012). Confinement of four radiotolerant species of the Bacillus class, elucidates its well documented radio-tolerance as they are resistant spore formers (Nicholson et al., 2000). The hydrolytic bacteria in our studies predominant and were from four distinct clusters or phyla with high survival rates at high dose of radiations. Other studies showed only -Proteobacteria that dominates in hydrolytic enzymes production in extreme environ (Rohban et al., 2009; Baati et al., 2010) where only a few of the Firmicutes were found. Isolation of Proteobacteria- related species in the waste contaminated with radio-isotopes of radionuclide were studied for its survival that exhibited resistance to 2.5 kGy of gamma radiation, with 0.0017% survival was given by Fredrickson and colleagues (2004).

The stimulation of bacterial growth with UVB exposure has already been reported on culturable bacteria and in total community analysis as well (Dib et al., 2009). The number of cells increases rapidly after exposure to UV and this increase in population and diversity under UVB stress need deeper studies in order to explain the mechanism triggered by radiations that enhances cellular survival and replication (McGlynn and Lloyd 2002). Most of the UVR isolates showed colored compounds when grown on TGY agar plates after the exposure to UV that may absorb radiation in order to protect the cells from damage. The production of UV absorbing compounds might be induced as a result of exposure to radiation stress. We have reported a UV resistant bacterium that has 91% similarity to Stenotrophomnas sp. representing an interesting

Page 153

phenomenon of enlargement in colony size and light pink to red coloration upon radiation exposure in presence of Mn+2. These secondary metabolite or extremolytes production during UVR exposure can be of high significance in pharmaceutical industry. Deinococcus genus is well known for their extreme resistance to UV radiation. Previously a number of studies have been carried out to isolate radio-resistant bacteria from desert soil, radiation resistant property is the result of evolution that protect cells from desiccation (Rainey et al., 2005). Recently it has been proposed that Deinococcus possesses a remarkable ability to cope with adverse conditions such as UV radiation and desiccation. This resistance is due to binding of the S-layer protein DR-2577 to deinoxanthin that could suggest its protective role against these two stresses (Farci et al., 2016). The ability of these UV resistant microbes to survive in several extreme conditions is suggested to be as a result of three combine mechanisms like prevention, tolerance and repair (White et al., , 1999).

The 09 UV resistant microbes were found to produce hydrolytic enzymes like amylase, DNase, protease, and were capable of growth at high salt concentration ranges from 2-16%. The results demonstrated a strong linkage between salt, metal and UV radiation tolerance. Metals like Mn+2, Cu+2, Zn+2 and Co+2 enhance the survivability of UVR microbes up to great extent These metals block the Fenton reactions and play an indirect role to prevent the formation of several toxic oxides and byproducts, which can alter the different cell membranes (Imlay 2008; Baati et al., 2010; Santos 2013). Responses to heavy metals tested in this study were very homogenous and all the strains isolated from both deserts showed high resistance to different metal ions with exception of Hg+2. This suggests that these UV resistant microbes express a very homogeneous behavior in connection with their individual natural resistance to different metal ions tested. The majority of the isolates showed resistant to Mn+2, Cr+2, Co+2, Ni+2 and Zn+2 which play an important role in the cell resistance to UV. The possible protective role of several metal ions against UV radiation has been discussed recently. Kineococcus radiotolerans (Actinobacteria) showed a high ability for intracellular copper ion sequestration that provided protection against the deleterious effects of ionizing radiation (Asgarani et al., 2012; Paulino-Lima et al., 2016). Manganese (Mn2+) ions also prevent oxidative damage instigated by several stresses encompassing UV-B radiation, gamma-irradiation, wet and dry heat and H2O2 (Daly et al., 2007; Barnese et al., 2008; McEwan, 2009; Daly, 2010; McNaughton, 2010; Slade, 2011). Adaptive response is mediated by the uptake of Zinc (Zn2+) which confers resistance against peroxide stress (Gaballa et al., 2002), thereby act as a barrier for copper-treated Escherichia coli against superoxide killing (Korbashi et al., 1989) and opposing the effects of oxidative stress in Lactococcus lactis (Scott et al., 2000). Sensitivity to Hg+2 by all isolates might be due to the fact that it has not been found to be essential for the UVR biological activities, furthermore the strong effect of Hg to cells relates to their strong affinity for thiol groups in proteins (Robinson et al 1994; Velasco et al., 1999). The ability of these microbes to grow in high UV radiation and high metal concentration make these more attractive for in-situ bioremediation of radioactive wastes.

Meticulous comprehension of the molecular effects of UVR on bacteria may pave the way for an indulgent of environmental repercussion of intense UV levels affiliated with global climate changes and will be followed by the optimization of UV-based disinfection strategies. Bacterial cell features such as small size,unavailability of effective UV-protective pigmentation and short generation time confers susceptibility to the effects of UV radiation, (Garcia-Pichel, 1994). The biological effects of UV radiation on all the resistant isolates were assayed that showed different survival rate and protein carbonylation and lipid peroxidation. The lipid and protein damages were analyzed by standard assays in UVB oxidative damages which are characterized by

Page 154

thiobarbituric acid reactive substances (TBARS) accumulation and loss of main metabolic activity. These UV damages also resulting in alteration in cell protein and lipids that ultimately hinder the growth or kill the cell. Deinococcus genra was considered the most resistant with less protein and lipid damages as compared to other isolates and E. coli (10536) ATCC used a control. The UV sensitive strain E. coli (10536) displayed significantly higher number of protein carbonyls than resistant isolates. The consequences associated with UVR exposure were determined by gas chromatography (Santos, 2013). An increase in the methyl groups was observed in the lipids upon its oxidation to UV. It is also investigated that change in the lipid chains and its composition occurs to the stress for survival.

In addition to DNA damages by high UV exposure changes in the lipid membranes and protein tertiary structure also have a crucial role in bacterial inactivation. The targets (e.g. nucleic acids, proteins, lipids) for UV radiation inactivation may differ among the genus, species and strains and thus all these factors contribute to the prolong cell survival in high UV radiations It has also been suggested that UV-induced DNA damage in Gram-positive bacteria is lower than that in Gram-negative bacteria because of a shielding effect by the cell wall (Jagger, 1985). Presence Mn/Fe ratio is another factor that can contribute to cell resistant in high radiations. Presence of high concentration of Fe+2 in Shewanella oneidensis MR1 makes it sensitive to UV radiations. These intracellular Fe promotes the formation of ROS via Fenton type reactions (Qiu et al., 2005). The efficiency of the defence and highly sophisticated molecular repair mechanisms, that may also differ among bacteria play an important role in cellular resistance in extreme environments (Arrieta et al., 2000; Matallana-Surget et al., 2009; Santos et al., 2011).

3.6. Conclusion

Analyzing and characterizing the culturable UV resistant bacteria from deserts, this study comes up with implications such as the discovery of a unique environment for UV resistant microbes in the context of Pakistan, production of UV absorbing compounds and investigation for heavy metal tolerant microorganisms. These results open the exiting standpoints on investigating bacterial lenience to desiccation, radiation and survey in the deserts. The implication of the outcomes is conferred from an environmental and industrial perspective and with admiration to potential expansions in UV-based disinfection technologies.

Page 155

References 1. Arrange, A.A., Phelps, T.J., Benoit, R.E., Palumbo, A.V. and White, D.C., 1993. Bacterial sensitivity to UV light as a model for ionizing radiation resistance. Journal of microbiological methods, 18(2), pp.127-136. 2. Arrieta, J.M., Weinbauer, M.G. and Herndl, G.J., 2000. Interspecific variability in sensitivity to UV radiation and subsequent recovery in selected isolates of marine bacteria. Applied and Environmental Microbiology, 66(4), pp.1468-1473. 3. Asgarani, E., Soudi, M.R., Borzooee, F. and Dabbagh, R., 2012. Radioresistance in psychrotrophic Kocuria sp. ASB 107 isolated from Ab-e-Siah radioactive spring. Journal of environmental radioactivity, 113, pp.171-176. 4. Baati, H., Amdouni, R., Gharsallah, N., Sghir, A. and Ammar, E., 2010. Isolation and characterization of moderately halophilic bacteria from Tunisian solar saltern. Current microbiology, 60(3), pp.157-161. 5. Bagwell, C.E., Bhat, S., Hawkins, G.M., Smith, B.W., Biswas, T., Hoover, T.R., Saunders, E., Han, C.S., Tsodikov, O.V. and Shimkets, L.J., 2008. Survival in nuclear waste, extreme resistance, and potential applications gleaned from the genome sequence of Kineococcus radiotolerans SRS30216. PloS one, 3(12), p.e3878. 6. Bagwell, C.E., Milliken, C.E., Ghoshroy, S. and Blom, D.A., 2008. Intracellular copper accumulation enhances the growth of Kineococcus radiotolerans during chronic irradiation. Applied and environmental microbiology, 74(5), pp.1376-1384. 7. Barnese, K., Gralla, E.B., Cabelli, D.E. and Selverstone Valentine, J., 2008. Manganous phosphate acts as a superoxide dismutase. Journal of the American Chemical Society, 130(14), pp.4604-4606. 8. Bligh, E.G. and Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Canadian journal of biochemistry and physiology, 37(8), pp.911-917. 9. Daly, M.J., Gaidamakova, E.K. and Matrosova, V.Y., 10. other authors (2004). Accumulation of Mn (II) in Deinococcus radiodurans facilitates gamma-radiation resistance. Science, 306, pp.1025-1028. 10. Daly, M.J., Gaidamakova, E.K., Matrosova, V.Y., Kiang, J.G., Fukumoto, R., Lee, D.Y., Wehr, N.B., Viteri, G.A., Berlett, B.S. and Levine, R.L., 2010. Small-molecule antioxidant proteome-shields in Deinococcus radiodurans. PloS one, 5(9), p.e12570. 11. Daly, M.J., Gaidamakova, E.K., Matrosova, V.Y., Vasilenko, A., Zhai, M., Leapman, R.D., Lai, B., Ravel, B., Li, S.M.W., Kemner, K.M. and Fredrickson, J.K., 2007. Protein

Page 156

oxidation implicated as the primary determinant of bacterial radioresistance. PLoS biology, 5(4), p.e92. 12. Dib, J.R., Weiss, A., Neumann, A., Ordoñez, O., Estévez, M.C. and Farías, M.E., 2009. Isolation of bacteria from remote high altitude Andean lakes able to grow in the presence of antibiotics. Recent patents on antiinfective drug discovery, 4(1), pp.66-76. 13. Farci, D., Slavov, C., Tramontano, E. and Piano, D., 2016. The S-layer Protein DR_2577 Binds Deinoxanthin and under Desiccation Conditions Protects against UV-Radiation in Deinococcus radiodurans. Frontiers in microbiology, 7. 14. Fleisher, M., Liu, T., Broecker, W.S. and Moore, W., 1999. A clue regarding the origin of rock varnish. Geophysical Research Letters, 26(1), pp.103-106. 15. Flores, M.R., Ordoñez, O.F., Maldonado, M.J. and Farías, M.E., 2009. Isolation of UV- B resistant bacteria from two high altitude Andean lakes (4,400 m) with saline and non saline conditions. The Journal of general and applied microbiology, 55(6), pp.447-458. 16. Fredrickson, J.K., Zachara, J.M., Balkwill, D.L., Kennedy, D., Shu-mei, W.L., Kostandarithes, H.M., Daly, M.J., Romine, M.F. and Brockman, F.J., 2004. Geomicrobiology of high-level nuclear waste-contaminated vadose sediments at the Hanford Site, Washington State. Applied and environmental microbiology, 70(7), pp.4230- 4241.

17. Gaballa, A. and Helmann, J.D., 2002. A peroxide induced zinc uptake system plays an important role in protection against oxidative stress in Bacillus subtilis. Molecular microbiology, 45(4), pp.997-1005. 18. Garcia Pichel, F., 1994. A model for internal self shading in planktonic organisms and its implications for the usefulness of ultraviolet sunscreens. Limnology and Oceanography, 39(7), pp.1704-1717. 19. Ghosh, S., Ramirez Peralta, A., Gaidamakova, E., Zhang, P., Li, Y.Q., Daly, M.J. and Setlow, P., 2011. Effects of Mn levels on resistance of Bacillus megaterium spores to heat, radiation and hydrogen peroxide. Journal of applied microbiology, 111(3), pp.663-670. 20. Halliwell, B. and Gutteridge, J.M., 2015. Free radicals in biology and medicine. Oxford University Press, USA. 21. Holm-Hansen, O., Lubin, D. and Helbling, E.W., 1993. Ultraviolet radiation and its effects on organisms in aquatic environments. In Environmental UV photobiology (pp. 379- 425). Springer US. 22. Imlay, J.A., 2008. Cellular defenses against superoxide and hydrogen peroxide. Annu. Rev. Biochem., 77, pp.755-776.

Page 157

23. Korbashi, P., Katzhendler, J., Saltman, P. and Chevion, M., 1989. Zinc protects Escherichia coli against copper-mediated paraquat-induced damage. Journal of Biological Chemistry, 264(15), pp.8479-8482. 24. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J biol Chem, 193(1), pp.265-275. 25. Macilwain, C., 1996. Science seeks weapons clean-up role. Nature, 383(6599), p.375. 26. Matallana-Surget, S., Douki, T., Cavicchioli, R. and Joux, F., 2009. Remarkable resistance to UVB of the marine bacterium Photobacterium angustum explained by an unexpected role of photolyase. Photochemical & Photobiological Sciences, 8(9), pp.1313- 1320. 27. Mattimore, V. and Battista, J.R., 1996. Radioresistance of Deinococcus radiodurans: functions necessary to survive ionizing radiation are also necessary to survive prolonged desiccation. Journal of bacteriology, 178(3), pp.633-637. 28. McCullough, J., Hazen, T. and Benson, S., 1999. Bioremediation of metals and radionuclides: what it is and how it works. Lawrence Berkeley National Laboratory. 29. McEwan, A.G., 2009. New insights into the protective effect of manganese against oxidative stress. Molecular microbiology, 72(4), pp.812-814. 30. McGlynn, P. and Lloyd, R.G., 2002. Genome stability and the processing of damaged replication forks by RecG. TRENDS in Genetics, 18(8), pp.413-419. 31. McNaughton, R.L., Reddi, A.R., Clement, M.H., Sharma, A., Barnese, K., Rosenfeld, L., Gralla, E.B., Valentine, J.S., Culotta, V.C. and Hoffman, B.M., 2010. Probing in vivo Mn2+ speciation and oxidative stress resistance in yeast cells with electron-nuclear double resonance spectroscopy. Proceedings of the National Academy of Sciences, 107(35), pp.15335-15339. 32. Misra, H.S., Khairnar, N.P., Barik, A., Indira Priyadarsini, K., Mohan, H. and Apte, S.K., 2004. Pyrroloquinoline quinone: a reactive oxygen species scavenger in bacteria. FEBS letters, 578(1-2), pp.26-30. 33. Moreno, M.L., Piubeli, F., Bonfa, M.R.L., García, M.T., Durrant, L.R. and Mellado, E., 2012. Analysis and characterization of cultivable extremophilic hydrolytic bacterial community in heavy metal contaminated soils from the Atacama Desert and their biotechnological potentials. Journal of applied microbiology, 113(3), pp.550-559.

Page 158

34. Nicholson, W.L., Munakata, N., Horneck, G., Melosh, H.J. and Setlow, P., 2000. Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiology and molecular biology reviews, 64(3), pp.548-572. 35. Office of Environmental Management, U.S. Department of Energy. (1996). 1996 Baseline environmental management report. Office of Environmental Management, U.S. Department of Energy, Washington, D.C. [Online.] http: //www.em.doe.gov/bemr96. 36. Pattison, D.I. and Davies, M.J., 2006. Actions of ultraviolet light on cellular structures. In Cancer: cell structures, and genomic instability (pp. 131-157). Birkhäuser Basel.

37. Paulino-Lima, I.G., Fujishima, K., Navarrete, J.U., Galante, D., Rodrigues, F., Azua- Bustos, A. and Rothschild, L.J., 2016. Extremely high UV-C radiation resistant microorganisms from desert environments with different manganese concentrations. Journal of Photochemistry and Photobiology B: Biology, 163, pp.327-336. 38. Pérez, J.M., Calderón, I.L., Arenas, F.A., Fuentes, D.E., Pradenas, G.A., Fuentes, E.L., Sandoval, J.M., Castro, M.E., Elías, A.O. and Vásquez, C.C., 2007. Bacterial toxicity of potassium tellurite: unveiling an ancient enigma. PLoS One, 2(2), p.e211. 39. Phillips, R.W., Wiegel, J., Berry, C.J., Fliermans, C., Peacock, A.D., White, D.C. and Shimkets, L.J., 2002. Kineococcus radiotolerans sp. nov., a radiation-resistant, Gram- positive bacterium. International journal of systematic and evolutionary microbiology, 52(3), pp.933-938. 40. Qiu, X., Sundin, G.W., Wu, L., Zhou, J. and Tiedje, J.M., 2005. Comparative analysis of differentially expressed genes in Shewanella oneidensis MR-1 following exposure to UVC, UVB, and UVA radiation. Journal of bacteriology, 187(10), pp.3556-3564. 41. Rainey, F.A., Ray, K., Ferreira, M., Gatz, B.Z., Nobre, M.F., Bagaley, D., Rash, B.A., Park, M.J., Earl, A.M., Shank, N.C. and Small, A.M., 2005. Extensive diversity of ionizing-radiation-resistant bacteria recovered from Sonoran Desert soil and description of nine new species of the genus Deinococcus obtained from a single soil sample. Applied and Environmental Microbiology, 71(9), pp.5225-5235. 42. Riley, R.G. and Zachara, J.M., 1992. Chemical contaminants on DOE lands and selection of contaminant mixtures for subsurface science research (No. DOE/ER--0547T). Pacific Northwest Lab., Richland, WA (United States). 43. Robinson, C.K., Webb, K., Kaur, A., Jaruga, P., Dizdaroglu, M., Baliga, N.S., Place, A. and DiRuggiero, J., 2011. A major role for nonenzymatic antioxidant processes in the

Page 159

radioresistance of Halobacterium salinarum. Journal of bacteriology, 193(7), pp.1653- 1662. 44. Robinson, J.B. and Tuovinen, O.H., 1984. Mechanisms of microbial resistance and detoxification of mercury and organomercury compounds: physiological, biochemical, and genetic analyses. Microbiological reviews, 48(2), p.95. 45. Rohban, R., Amoozegar, M.A. and Ventosa, A., 2009. Screening and isolation of halophilic bacteria producing extracellular hydrolyses from Howz Soltan Lake, Iran. Journal of industrial microbiology & biotechnology, 36(3), pp.333-340. 46. Santos, A.L., Gomes, N., Henriques, I., Almeida, A., Correia, A. and Cunha, A., 2013. Role of Transition Metals in UV B Induced Damage to Bacteria. Photochemistry and photobiology, 89(3), pp.640-648. 47. Santos, A.L., Lopes, S., Baptista, I., Henriques, I., Gomes, N.C.M., Almeida, A., Correia, A. and Cunha, A., 2011. Diversity in UV sensitivity and recovery potential among bacterioneuston and bacterioplankton isolates. Letters in applied microbiology, 52(4), pp.360-366. 48. Santos, A.L., Moreirinha, C., Lopes, D., Esteves, A.C., Henriques, I., Almeida, A., Domingues, M.R.M., Delgadillo, I., Correia, A. and Cunha, A., 2013. Effects of UV radiation on the lipids and proteins of bacteria studied by mid-infrared spectroscopy. Environmental science & technology, 47(12), pp.6306-6315. 49. Santos, A.L., Oliveira, V., Baptista, I., Henriques, I., Gomes, N.C., Almeida, A., Correia, A. and Cunha, Â., 2013. Wavelength dependence of biological damage induced by UV radiation on bacteria. Archives of microbiology, 195(1), pp.63-74. 50. Scott, C., Rawsthorne, H., Upadhyay, M., Shearman, C.A., Gasson, M.J., Guest, J.R. and Green, J., 2000. Zinc uptake, oxidative stress and the FNRlike proteins of Lactococcus lactis. FEMS microbiology letters, 192(1), pp.85- 89. 51. Singh, O.V. and Gabani, P., 2011. Extremophiles: radiation resistance microbial reserves and therapeutic implications. Journal of applied microbiology, 110(4), pp.851-861. 52. Slade, D. and Radman, M., 2011. Oxidative stress resistance in Deinococcus radiodurans. Microbiology and Molecular Biology Reviews, 75(1), pp.133191.

53. Tamura, K. and Nei, M., 1993. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular biology and evolution, 10(3), pp.512-526.

Page 160

54. Tamura, K., Stecher, G., Peterson, D., Filipski, A. and Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular biology and evolution, 30(12), pp.2725-2729. 55. Velasco, A., Acebo, P., Flores, N. and Perera, J., 1999. The mer operon of the acidophilic bacterium Thiobacillus T3. 2 diverges from its Thiobacillus ferrooxidans counterpart. Extremophiles, 3(1), pp.35-43. 56. White, O., Eisen, J.A., Heidelberg, J.F., Hickey, E.K., Peterson, J.D., Dodson, R.J., Haft, D.H., Gwinn, M.L., Nelson, W.C., Richardson, D.L. and Moffat, K.S., 1999. Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1. Science, 286(5444), pp.1571-1577.

Chapter 4: Extremolytes Extraction and Bioassays

Page 161

Paper 2

Title: In-Vitro Cytotoxic and Antioxidant Activities of Extremolytes from RadioResistant Bacteria

Page 162

4.1. Abstract Historically the study of microbial driven natural product as drug has largely ignored the radio-resistant extremophiles. From the limited courtesy received, many compounds of extremophilic origin have been described that displayed anticancer drugs, cholesterol-lowering agents, antibacterial activities and tools for research across a range of potencies. The secondary intracellular bio-active compounds isolated from radio resistant microbes were assayed for cytotoxic, antioxidant and antibacterial activities to evaluate their potential to be considered as therapeutic drugs. The methanolic extracts of these radio-resistant isolates were also scanned for it excitation wavelength using photo luminescence spectroscopy. 2,2-diphenyl-1picrylhydrazyl (DPPH) reducing assay, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using HeLa cell line and the disc diffusion test was carried out in order to evaluate the partially purified crude extracts. A pronounced cytotoxic effect on HeLa cell lines and anti-oxidant activity was shown by Deinococcus WMA-LM9 (50 µg). Similarly, the high anti-bacterial activity was detected in case of extract from Stenotrophomonas sp. WMA-LM19 against N. gonorrhoea MS11 and Rhodococcus sp. The current research work concluded that isolated strains from extreme environment have great potential to produce potent metabolites with a wide range of anti- oxidant and cytotoxic activities. Exploration of these microbes and metabolites are basic need for new drug discovery.

Keywords: Radio-resistant, Deinococcus WMA-LM9, Stenotrophomonas sp. WMALM19, DPPH assay, MTT assay, Anti-bacterial assay

4.2. Introduction Ionizing radiation, distinctly, the low linear energy transfer (LET), happens to destroy the vital macromolecules like protein structures, lipid membranes, DNA and RNA of the target cell by generating free radicals. Cellular targets, i.e. macromolecules, suspended in the biological aqueous environment react with high energy free radicals and become ionized or convert into toxic free radicals (Halliwell et al., 2015). These types of chemical alterations lead to transformed cell metabolism, structural and functional damage and cell death by disrupting molecular organization (Goodson et al., 2015). Radiation-induced disruptions in DNA includes damaged pyrimidine and purine bases, cross-linking of DNA with adjacent protein molecules, removal of bases and single and double strand break (Rastogi et al., 2010). The most adverse effect of irradiation in general is on lipid as lipid peroxidation in all biological membranes while mitochondrial membrane in particular. Lipid peroxidation leads to the formation of short chain fatty acyl derivatives, which interfere in lipid-lipid as well as lipid-protein cross-linkages formation (Pizzimenti et al., 2013). This type of interference encourages drastic deformity in biological functions also protein denaturation and cleavages of disulphide bond in protein and oxidation of

Page 163

approachable amino acids leads to oxidative stress. All these changing leads to membrane permeability and fluidity which trigger the release of effective physiological mediators of apoptosis (Upadhyay et al., 2005). These stresses induced protein and lipid modifications leads to premature aging cancer.

The radio-resistant microbes are supposed to have a high efficiency system for proteome protection as compared to genome (Santos et al., 2013). Proteome is believed to ensure recovery of cell from adverse radiation effects by molecular repair, followed by repair of disintegrated DNA, that’s the reason why death correspond with protein damage instead of DNA by irradiation. The proteome conserves and maintain life while genome makes sure the uniformity of life by renewing proteins. The process depends on the preceding proteome that restore, replicate and expressed genome. Moreover, other small metabolites cofactors for protein interaction and catalysis are of equivalent importance of proteome functionality however the role of protein damage on conservation of life is underated in biology and medicine (Krisko et al., 2013). However, considering above fact, many therapeutic agents, having ability to rescue organisms from radiation injury has been discovered (Ghose, 1983; Nair et al., 2004). Many health benefits are associated with biological activities of carotenoids and other bio-active compounds like antioxidants and precursors for vitamin A biosynthesis (Grune et al., 2010). The carotenoid rich dietary intake is associated with enhancement of the immunity and devaluation of liability for degenerative diseases such as cancer, cardiovascular defects, macular degeneration, and cataract (Krinsky et al., 2000; Fiedor et al., 2014). These benefits diversified the novel functionalities of carotenoids in foods, cosmetics, and pharmaceutical applications. A monumental documentation available about the expression of secondary metabolite genes under stress conditions. The transformation of a single variable or factor and provoking a stress response in growth conditions of microbes has already been investigated in one strain many compounds (OSMAC) approaches for the exploration the possibility of secondary metabolites through various strains of cyanobacteria (Edwards and Ericsson, 1999). A group of oxigenases (hemoprotein) like Cytochrome P450s CYPs are ubiquitous present in all domain of life (Nebert et al., 1989). Modern biotechnological approaches could be used to activate the induction of the biosynthesis of radiation responsive pigments/bio-active compounds and cellular metabolites which can be utilized by other organisms to provide protection to live in a radiation rich environment (Singh, 2011). In UV-light, UV-resistant microorganisms can be grown in these conditions leads to induction of genes that produce metabolites which defensive against harmful radiations. It has been expected that useful drugs exclusively anticancer and antibiotics and further agricultural products of commercial implications can be obtained these compounds i.e extremolytes (Kumar et al., 2010). The Deinococcus radiodurans is well-documented radio-resistant microorganism, its survival lies in the fact that it is able to induce certain genes, proteins and DNA repair mechanism enzyme (Hockberger, 2002).

Development has been made to search extremophiles having ability to produce extremolytes with an indication as productive as well as therapeutic agents. The modern biotechnological techniques and biosynthesis of radiation responsive pigments or bio-active compounds could be enhanced to furnish a chance for other organisms to sustain radiation rich surroundings (Singh and Gabani, 2011). Numerous extracts (i.e. extremolytes) are known to yield vital drugs, specifically antibiotics and anticancer drugs (Kumar et al., 2010). These extremolytes are of prime importance for industrial use as it has been investigated, however, therapeutic intimations are remade to investigate. The challenges for further research include the

Page 164

implementation of these UVR resistant antimicrobial, anti-oxidant is to prevent aging, DNA mutation and cancer related protein modifications (Slade and Radman, 2011).

A comprehensive review has been made on bioactive compound derivatives from ultra violet radiations (UVR) resistant bacteria. Their remarkable biological properties as antimicrobial, cytotoxic activities using HeLa cell lines and detailed antioxidant system in terms of hydroxyl radicals scavenging for toxic superoxide’s was studied.

4.3. Materials and Methods

4.3. Materials and methods The radio-resistant bacteria were isolated from Lakki Marwat and Bahawalpur desert soil of Pakistan. Isolates were identified on the basis of method described in Bergey’s Manual of Systematic Bacteriology (Buchanan and Gibbons, 1974; Wilson, 1987) and phylogenetic analysis was done on the basis of 16S rRNA sequence (Marchesi et al. 1998).

4.3.1. Growth Medium The isolates were grown in tryptone glucose yeast (TGY) medium (5% Bactotryptone, 1% glucose, 3% Bacto yeast extract) and incubated in shaking incubator at 30°C with 150 rpm for 72 hrs.

4.3.2. Collection of Intracellular Crude Extracts 500 mL of each UVR resistant bacterial isolates were grown with steady shaking under aerobic conditions. Cells were collected after 40hrs of incubation by centrifugation at 5000xg for 10 min. The cells in sediment were used to collect intracellular extract. Once washed with sterilized water, the cells were re-suspended in 5 mL chloroform:methanol (7:3), probe sonicated and then washed with 100% dichloromethane to ensure extraction process, finally the mixture is centrifuged. The supernatant with solvent mixture was evaporated and the dried intracellular extract was collected on freeze drying. The dried crude extract was then again put through solid phase extraction using 500 mg of C18 cartridge with methanol/water (50:50 v/v) acetone:methanol (7:2 v⁄v) and dichloromethane and methanol (2:1).

4.3.3. Excitation vs Emission Spectra by using Photoluminescence Spectroscopy Photoluminescence spectroscopy is a non-destructive method that is concerned with transitions of photons from the excited state to the ground state. Fluorescence spectra of methanolic extract were taken on an LS-50B fluorescence spectrometer (Perkin-Elmer Corp., Forster City, CA, USA) with an external 980 nm laser as excitation source. The spectrum range used (300–900 nm) with a double beam Fluorescent using a 5-cm path quartz cuvette against methanol as a reference.

4.3.4. Sample Preparation for Biological Assays The intracellular crude extract was dispersed in dimethyl sulphoxide (10mg/mL DMSO), protected from light and stored at room temperature. For the cytotoxic assay, the extract volume was set in such a way that the final concentration of DMSO does not exceed 0.1%.

Page 165

4.3.5. DPPH Radical Scavenging Activity The antioxidant ability of the extracts was evaluated by a standard procedure method described by Rao et al., (2006), using 2,2-diphenyl-1-picrylhydrazyl (DPPH). Different concentrations of the partially purified extract (ranges from 5-20µg) were added to ethanolic solution of DPPH (0.2mM), only DMSO was mixed in case of control. The reaction mixture was then vortexed and incubated for 30 min at room temperature under dark condition. The absorbance of the reaction mixture was measured at 517 nm using spectrophotometer. The scavenging capacity of the extracts was measured by a decrease in the absorbance of DPPH (a negative control). Ascobic acid was run as positive control. 4.3.6. Cytotoxic Evaluation of Crude Extracts In vitro tests were performed on HeLa cells to check cytotoxic effect of extract using a rapid colorimetric assay with MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5Diphenyltetrazolium Bromide) and untreated controls were also present to compare results. The HeLa cells were purchased from ATCC by Mark Leid's lab (Department of Pharmaceutical Sciences, Oregon State University) cultured in RPMI-1640 (Roswell Park Memorial Institute) (Gibco, Scotland) in 75 cm2 tissue flask (Nunc, Denmark), cultured in the T75 flask with MEM (minimal essential medium) 10% FBS (fetal bovine serum) and 1% pen/step (penicillin/streptomycin). The assay is based on the reduction potential of the soluble MTT of tumor cells by the mitochondrial enzyme action (2000 and 10,000 cells/well), into an insoluble product called formazan. Which can be measured spectrophotometrically after dissolving in DMSO (Denizot and Lang 1986). The cytotoxic effect of the extract was indicated as relative viability (% control) and is calculated. Percentage of the cell survival in negative control was presumed as 100% (Ngeny et al., 2013).

4.3.7. Evaluation of Antimicrobial Activity Analysis of antimicrobial activity of extracts was assayed by the agar diffusion test against clinically and ATCC strains on agar plates. Neisseria gonorrhoea MS11 (Meyer et al., 1982) strains were cultured on gonococcal base (GCB) solid medium (Difco) augmented with Kellogg’s supplements I and II (Kellogg et al 1982; Spence et al., 2008) at

37°C, in the presence of 5% atmospheric CO2 for 20 hours. Nonpalliated variants of bacterial colonies were sub cultured in fresh GCB medium for additional 20 hours. Vibrio cholera N16961 (laboratory collection) was cultured on LB agar (Difco) plates medium at 37°C. Rhodococcus fascians ATCC 12975, S. aureus ATCC 6538, E. coli ATCC 11303 and Pseudomonas aeruginosa ATCC 10145 were cultured on Muller Hinton agar (MHA) plates. The antibacterial activity was analyzed by following standard guidelines of Clinical and Laboratory Standard Institute (CLSI) by using disk diffusion method (Schwalbe et al., 2007; Mbaveng et al., 2008). Antimicrobial activity was determined by measuring zones of inhibition in mm and the results recorded in triplicate. The extract fractions with zone of inhibition more than 10 mm were considered to be active.

4.3.8. Statistical analysis Mean, standard deviation (SD) and coefficient of variation (CV) were determined, for concentrations of each drug. Significance was presumed at p<0.05.

Page 166

4.4. Results 4.4.1. Solid Phase Extraction of intracellular metabolites Once the intracellular metabolites extracted in a solvent mixture, dried and further fractionated to 3 fractions in water: methanol, acetone/methanol and dichloromethane vacuum evaporated and dissolved in DMSO for further studies.

4.4.2. Solid Phase Extraction of Intracellular Metabolites Once the intracellular metabolites extracted in a solvent mixture, dried and further fractionated to 3 fractions in water:methanol, acetone:methanol and dichloromethane vacuum evaporated and dissolved in DMSO for further studies.

4.4.3. Excitation of Methanolic Extract Photo-excitation aids electrons within a material to move into legitimate excited states. Extracts of Deinococcus sp. WMA-LM9 showed a greater excitation state followed by Kocuria sp. WMA-BD1 and Deinococcus sp. WMA-LM30. The excitation is due to the colored part or chromophore of these compounds. All the compounds extracted from these UV resistant microbes showed a peak of photoluminescence at wavelength ranges from 400-600 nm as shown in figure 4.1.

.

Figure 4.1: The fluorescent quenching spectrum of methanolic extracts 1mg/ml from UVR bacteria with an excitation wavelength of 316 nm. The data was plotted by using Origin 8.6 software.

Page 167

4.4.3. Cytotoxic activity of the crude extracts MTT staining method revealed about the cytotoxic effect of different metabolites extracted from all isolates. Our results demonstrated that the extracts of 5 isolates had strong effects on HeLa cells mostly in a time-dependent manner (Fig. 4.2). The most remarkable activity against HeLa cell lines was shown by methanolic fraction of Stenotrophomonas sp. WMA-LM19, Deinococcus sp. WMA-LM9 and Kocuria sp.

WMA-BD1 and showed 90% killing of HeLa cells when used in concentrations of 50 µg.

4.4.4. Comparative IC50 Values of Extracts by Anti-oxidant Assay The DPPH radical scavenging activity of different extracts was measured as shown in figure 4.3. The scavenging activity was found to be concentration dependent. Deinococcus sp. WMA-LM9 (F2; methanolic fraction) and Deinococcus sp. WMA-LM30 (dichloromethane extract) was the most potent one with having IC50 of 10 µg. IC50 was acquired by linear regression analysis of dose response curve that has been plotted between % inhibition and carotenoid concentration that led to 50% inhibition of free radical activity of DPPH.

4.4.5. Anti-microbial Activity of Fractionated Extracts Significant anti-microbial activity against N. gonorrhoea MS11, E. coli ATCC 11303, P. aeruginosa ATCC 10145, S. aureus ATCC 6538 and R. fascians ATCC 12975 by UV resistant microbial organic and aqueous extracts. The highest activity was achieved by WMA-LM19-F1 and F2 against N. gonorrhoea MS11 and R. fascians. ATCC 12975 (25mm) followed by WMA-LM9-F2 (22 mm and 18 mm) respectively

(table 4.1 and figure 4.4).

Page 168

Figure 4.2: The cytotoxic effect of partially purified fractionated extracts on HeLa cell line by using MTT assay. The various concentrations of extracts used were 50 µg/mL. The data represent the percentage (%) inhibition. Values are expressed as mean ± SD (n=3). F1= Water:methanolic fraction, F2= acetone:methanolic extract and F3= Dichloromethane fraction.

Page 169

Figure 4.3: Comparative analysis of IC50 values of different fractionated extracts of UV resistant microbes. IC50 of microbial extracts used for all the activities are measured in µg/ml. Data is expressed as mean ± SD (n=3). *p< 0.0001 vs 0 µg/ml.

Figure 4.4: Anti-microbial assay of partially purified extracts from radio resistant bacteria. Each extracts were used at the concentration of 10µg and zone of inhibitions were measured in mm after 18 hours of incubation at 37 °C. The values are mean ± standard deviation (n=3). (tet = tetracycline, car= carbencillin, pen= penicillin, amp= ampicillin).

Table. 4.2: Anti-microbial activity of partially purified extracts from UV resistant microbes. Diameter of the inhibitory zones recorded in mm. n = 3, Values mean ± SEM.

S.no Crude extracts S. E. P. N. R. aureus coli aeurogenosa gonorrhoeae fascians 6538 11303 10145 MS11 12975

1 WMA-BD2-F1 11 NZ NZ NZ 11

2 WMA-LM4-F1 8 14 NZ NZ NZ

3 WMA-LM9-F2 12 NZ NZ 22 18

4 WMA-LM19-F1 12 10 12 15 25

5 WMA-LM19-F2 NZ NZ NZ 25 NZ

Page 170

6 WMA-LM30-F2 NZ NZ NZ 8 14

WMA-BD1-F1 NZ NZ NZ 22 NZ 7

8 PC 28 20 20 20 8

F1=Water:Methanol, F2=Methanolic and F3=Dichloromethane fraction. NZ= No zone, PC= Positive control

4.5. Discussion:

The radiations induced oxidative damage is a versatile phenomenon which affects the regulation of key cellular processes such as proliferation, repair and recovery (Szumiel et al., 2015). Reactive nitrogen/oxygen species (RNS/ROS) lead to DNA fragmentation, membrane damages and lesions. In current study, partially purified extracts from UVR resistant bacterial strains that have been isolated from two different deserts of Pakistan were assessed for their cytotoxic, antioxidant and antibacterial activities. Several biochemical methods like free radical scavenging properties by using DPPH assay, cytotoxic assay by using HeLa cell lines, antibacterial assay by disc diffusion method were performed.

The photoluminescence spectra of methanolic extracts were recorded showing that all the compounds seem to show a high peak of excitation at wavelengths ranging from 400-700. It indicates that all extracts from radio-resistant isolates have UV absorbing potential. The fluorescence of these UV absorbing compounds is the result of photons being emitted (vibrational energy), as molecules come to ground state from excited state’s Comparatively fastest mechanism is followed as molecule returns to their ground state, fluorescence can only be observed when there are more systematic ways of relaxation (Hodak and Valeur, 2008). Photo- resistance can be achieved by quenching the energy in an excited state from UV absorbing compounds and coupling it with photosensitive cells or tissues. Photoluminescence is associated with the energy level differences between two electron states that are being in transition between excited and the equilibrium state (Valeur et al., 2012). In most of the photo luminescent systems, the chromophore aggregation mostly quenches light emission via aggregation caused quenching (Yuan et al., 2010).

High production of various super oxide and nitric oxide radicals confers to the pathogenesis of some inflammatory diseases and other pathological conditions (Guo et al., 1999).

Page 171

Current research shows that the different metabolic extracts from radioresistant bacteria can effectively quench DPPH, a free radical containing reactive oxygen/nitrogen species (ROS/RNS) by using DPPH assays. In in-vitro system extracts that were obtained from UV resistant bacteria are used to inhibit oxide radical pathways and remarkable inhibition is reported. Compounds from these UVR resistant bacteria may be contemplated with their same antioxidant activity under radiation-induced pathological processes. The reaction between bioactive compounds having free radicals at their conjugated double bonds following additional reaction, leads to the formation of stable products that keep the cell tissues from significant damage (Krinsky et al., 2005). Our investigations showed the scavenging abilities of extremolytes against non-site-specific hydroxyl ion from the UVR resistant microbes.

The ability of the extracts from UV resistant bacteria to exhibit cytotoxic activity against HeLa cells and antimicrobial activity against some of the bacteria suggested the presence of hydrophobic and hydrophilic bioactive compounds. Previous studies on preliminary screening of extracts from bacteria and fungi revealed the presence of saponins, triterpenoids and the flavonoids but no alkaloids (Ellithey et al., 2013). The results obtained from this study indicate that most of the microbial extracts from UV resistant microbes have displayed significant antibacterial and cytotoxic activity. MTT assays to predict the cytotoxic effects that targets the activity of in mitochondria reducing tetrazolium salt into formazan crystals (Duh et al., 1998). The color intensity of formazan dye corresponds to the sum of potent cells. The MTT assay evaluates the activity cell on the bases of generation of reducing equivalents in metabolically active cells (Cartuche et al., 2015).

Not much research has been done on the anticancer activities of Deinococcus UV resistant genera. We reported 5 potent and high priorities hits as an anti-cancerous C18 partially purified compounds. The extracts from Kocuria, Deinococcus and Stenoptrophomonas species were found to have high cytotoxic activity against cell lines of cancer and perhaps the potential candidates for the isolation of bioactive molecules. As far as we know, no report is available concerning the isolation of bioactive molecules that have cytotoxic activity, from Stenoptrophomonass, Deinococcus and Kocuria sp. These bioactive compounds with significant cytotoxic activity with cancer cell lines might be very useful as antitumor, anti-proliferative and other bioactive agents. The present study showed that different fractions of the cellular extract were active against E. coli ATCC11303, S. aureus ATCC 6538, R. fascians ATCC 12975 (a plant pathogen) and most importantly drug resistant N. gonorrhoea MS11. Di-terpenoid and phenolic compounds are hydrophobic in nature, thus targets the cytoplasmic membrane and preferentially create partition into the lipid bilayer (Kyrikou et al., 2005). The results of our antibacterial study showed that the extract from Stenotrophomons sp. WMA-LM19 and Deinococcus sp. WMA-LM9 showed better inhibitory activity against N. gonorrhoea MS11 and other gram positive and negative bacteria in comparison to other antibiotics.

4.6. Conclusion Some of the extracts showed good antioxidant, cytotoxic and antibacterial activities. Our results suggested that these UVR resistant microbes can be a potential source of new therapeutic drugs and metabolite isolation in the field of biotechnology. To our knowledge this is the first report of anti- N. gonorrhoea and R. fascians12975 (plant pathogen) activities of bio active compounds isolated from a UV resistant Stenoptrophomonas sp. and other isolates. In this study, we also investigated that strain Stenotrophomonas sp. WMA-LM19 and Deinococcussp. WMA-LM9

Page 172

not only able to survive in high UV doses, H2O2 and Mitomycin C but can also produce highly active and UV absorbing compounds. These two strains were selected for further studies.

Acknowledgment

This work was supported by grants from Higher Education Commission of Pakistan under international research support initiative program (IRSIP). We also highly acknowledge Oregon State University for providing the opportunity to work in collaboration.

Conflict of interest

No conflict of interest is associated with this work.

References

1. Bendich, A., 2001. Dietary reference intakes for vitamin C, vitamin E, selenium, and carotenoids institute of medicine washington, DC: National Academy Press, 2000 ISBN: 0- 309-06935-1. Nutrition, 17(4), p.364. 2. Buchanan, R.E., 1974. Bergey's manual of determinative bacteriology (No. 589.9 B47 1974). 3. Cartuche, L., Cruz, D., Ramírez, M.I., Bailón, N. and Malagón, O., 2015. Antibacterial and cytotoxic activity from the extract and fractions of a marine derived bacterium from the Streptomyces genus. Pharmaceutical biology, 53(12), pp.1826-1830. 4. Denizot, F. and Lang, R., 1986. Rapid colorimetric assay for cell growth and survival: modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. Journal of immunological methods, 89(2), pp.271277. 5. Duh, C.Y., Wang, S.K., Chu, M.J. and Sheu, J.H., 1998. Cytotoxic sterols from the soft coral Nephthea erecta. Journal of natural products, 61(8), pp.1022-1024. 6. Edwards, P.A. and Ericsson, J., 1999. Sterols and isoprenoids: signaling molecules derived from the cholesterol biosynthetic pathway. Annual review of biochemistry, 68(1), pp.157-185.

Page 173

7. Ellithey, M.S., Lall, N., Hussein, A.A. and Meyer, D., 2013. Cytotoxic, cytostatic and HIV- 1 PR inhibitory activities of the soft coral Litophyton arboreum. Marine drugs, 11(12), pp.4917-4936. 8. Fiedor, J. and Burda, K., 2014. Potential role of carotenoids as antioxidants in human health and disease. Nutrients, 6(2), pp. 466-488. 9. Ghose, A., 1983. Protection with combinations of hydroxytryptophan and some thiol compounds against whole-body gamma irradiation. International Journal of Radiation Biology and Related Studies in Physics, Chemistry and Medicine, 44(2), pp.175-181. 10. Goodson, W.H., Lowe, L., Carpenter,

D.O., Gilbertson, M., Manaf Ali, A.,

Lopez de Cerain Salsamendi, A.,

Lasfar, A., Carnero, A., Azqueta, A.,

Amedei, A. and Charles, A.K., 2015. Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: the challenge ahead. Carcinogenesis, 36(Suppl_1), pp.S254-S296. 11. Grune, T., Lietz, G., Palou, A., Ross, A.C., Stahl, W., Tang, G., Thurnham, D., Yin, S.A. and Biesalski, H.K., 2010. β-Carotene is an important vitamin A source for humans. The Journal of nutrition, 140(12), pp.2268S-2285S. 12. Guo, X., Wang, W.P., Ko, J.K. and Cho, C.H., 1999. Involvement of neutrophils and free radicals in the potentiating effects of passive cigarette smoking on inflammatory bowel disease in rats. Gastroenterology, 117(4), pp.884-892. 13. Halliwell, B. and Gutteridge, J.M., 2015. Free radicals in biology and medicine. Oxford University Press, USA. 14. Hockberger, P.E., 2002. A history of ultraviolet photobiology for humans, animals and microorganisms. Photochemistry and photobiology, 76(6), pp.561-579. 15. Hodak, J., Harvey, D. and Valeur, B., 2008. Introduction to fluorescence and phosphorescence. Spectroscopy for Chemistry BSAC program.

Page 174

16. Kellogg, D.S., Peacock, W.L., Deacon, W.E., Brown, L. and Pirkle, C.I., 1963. Neisseria gonorrhoeae I. Virulence genetically linked to clonal variation. Journal of Bacteriology, 85(6), pp.1274-1279. 17. Krinsky, N.I. and Johnson, E.J., 2005. Carotenoid actions and their relation to health and disease. Molecular aspects of medicine, 26(6), pp.459-516. 18. Krisko, A. and Radman, M., 2013. Biology of extreme radiation resistance: the way of Deinococcus radiodurans. Cold Spring Harbor perspectives in biology, 5(7), p.a012765. 19. Kumar, R., Patel, D.D., Bansal, D.D., Mishra, S., Mohammed, A., Arora, R., Sharma, A., Sharma, R.K. and Tripathi, R.P., 2010. Extremophiles: sustainable resource of natural compounds-extremolytes. In Sustainable Biotechnology (pp. 279-294). Springer Netherlands. 20. Kyrikou, I., Georgopoulos, A.,

Hatziantoniou, S., Mavromoustakos,

T. and Demetzos, C., 2005. A comparative

study of the effects of cholesterol

and sclareol, a bioactive labdane type diterpene, on phospholipid bilayers. Chemistry and physics of lipids, 133(2), pp.125-134.

21. Marchesi, J.R., Sato, T., Weightman, A.J., Martin, T.A., Fry, J.C., Hiom, S.J. and Wade, W.G., 1998. Design and evaluation of useful bacteriumspecific PCR primers that amplify genes coding for bacterial 16S rRNA. Applied and environmental microbiology, 64(2), pp.795-799.

Page 175

22. Mbaveng, A.T., Ngameni, B., Kuete,

V., Simo, I.K., Ambassa, P., Roy, R.,

Bezabih, M., Etoa, F.X., Ngadjui, B.T.,

Abegaz, B.M. and Meyer, J.M., 2008.

Antimicrobial activity of the crude extracts and five flavonoids from the twigs of Dorstenia barteri (Moraceae). Journal of

Ethnopharmacology, 116(3), pp.483-489. 23. Meyer, T.F., Mlawer, N. and So, M., 1982. Pilus expression in Neisseria gonorrhoeae involves chromosomal rearrangement. Cell, 30(1), pp.45-52. 24. Nair, C.K.K., Salvi, V., Kagiya, T.V. and Rajagopalan, R., 2004. Relevance of radioprotectors in radiotherapy: studies with tocopherol monoglucoside. Journal of environmental pathology, toxicology and oncology, 23(2). 25. Nebert, D.W., Nelson, D.R., Adesnik, M., Coon, M.J., Estabrook, R.W., Gonzalez, F.J., Guengerich, F.P., Gunsalus, I.C., Johnson, E.F., Kemper, B. And Levin, W., 1989. The P450 superfamily: updated listing of all genes and recommended nomenclature for the chromosomal loci. Dna, 8(1), pp.1-13. 26. Ngeny, L.C., Magiri, E., Mutai, C., Mwikwabe, N. and Bii, C., 2013. Antimicrobial properties and toxicity of Hagenia abyssinica (Bruce) JF Gmel, Fuerstia africana TCE Fries, Asparagus racemosus (Willd.) and Ekebergia capensis Sparrm. African Journal of Pharmacology and Therapeutics, 2(3). 27. Pizzimenti, S., Ciamporcero, E.S., Daga, M., Pettazzoni, P., Arcaro, A.,

Cetrangolo, G., Minelli, R., Dianzani, C., Lepore, A., Gentile, F. and Barrera, G., 2013. Interaction of aldehydes derived from lipid peroxidation and membrane proteins. Frontiers in physiology, 4, p.242.

Page 176

28. Rao, A.R., Sarada, R., Baskaran, V. and Ravishankar, G.A., 2006. Antioxidant activity of Botryococcus braunii extract elucidated in vitro models. Journal of Agricultural and Food Chemistry, 54(13), pp.4593-4599. 29. Rastogi, R.P., Kumar, A., Tyagi, M.B. and Sinha, R.P., 2010. Molecular mechanisms of ultraviolet radiation-induced DNA damage and repair. Journal of nucleic acids, 2010. 30. Santos, A.L., Oliveira, V., Baptista, I., Henriques, I., Gomes, N.C., Almeida, A., Correia, A. and Cunha, Â., 2013. Wavelength dependence of biological damage induced by UV radiation on bacteria. Archives of microbiology, 195(1), pp.63-74. 31. Schwalbe, R., Steele-Moore, L., and Goodwin, A.C., 2007. Antimicrobial susceptibility testing protocols. Crc Press. 32. Singh, O.V. and Gabani, P., 2011. Extremophiles: radiation resistance microbial reserves and therapeutic implications. Journal of applied microbiology, 110(4), pp.851-861. 33. Slade, D. and Radman, M., 2011. Oxidative stress resistance in Deinococcus radiodurans. Microbiology and Molecular Biology Reviews, 75(1), pp.133191. 34. Spence, J.M., Wright, L. and Clark, V.L., 2008. Laboratory maintenance of Neisseria gonorrhoeae. Current protocols in microbiology, pp.4A-1. 35. Szumiel, I., 2015. Ionizing radiation-induced oxidative stress, epigenetic changes and genomic instability: the pivotal role of mitochondria. International journal of radiation biology, 91(1), pp.1-12. 36. Upadhyay, S.N., Dwarakanath, B.S., Ravindranath, T. and Mathew, T.L., 2005. Chemical radioprotectors. Defence Science Journal, 55(4), p.403. 37. Valeur, B. and Berberan-Santos, M.N., 2012. Molecular fluorescence: principles and applications. John Wiley & Sons. 38. Wilson, K., 1987. Preparation of genomic DNA from bacteria. Current protocols in molecular biology, pp.2-4. 39. Yuan, W.Z., Lu, P., Chen, S., Lam, J.W., Wang, Z., Liu, Y., Kwok, H.S.,

Ma, Y. and Tang, B.Z., 2010. Changing the Behavior of Chromophores from Aggregation Caused Quenching to Aggregation Induced Emission:

Development of Highly Efficient Light Emitters in the Solid State. Advanced materials, 22(19), pp.2159-2163.

Page 177

Chapter 5: Radio-protective and Antioxidative Activities of Astaxanthin

Paper 3

Title: Radio-protective and Antioxidative Activities of Astaxanthin from Newly Isolated Radio-resistant Bacterium Deinococcus sp. Strain WMA-LM9

Published in Annals of Microbiology

Page 178

5.1. Abstract A radio-resistant bacterium, designated as strain WMA-LM9, was isolated from desert soil. 16S rRNA gene sequencing indicated that the bacterium belongs to genus Deinococcus with maximum similarity to Deinococcus radiopugnans. Deinococcus sp. strain WMA-LM9 was found to be resistant to an ultraviolet (UV) dose of 5 × 103 J/m2, hydrogen peroxide (50 mM) and mitomycin C (10 μg/ml). A carotenoid pigment was extracted using chloroform/methanol/acetone (7:5:3) and purified by high-performance liquid chromatography on a C18 analytical column. The compound was characterized as mono-esterified astaxanthin by 1H, 13C nuclear magnetic resonance and mass spectrometry. It was tested for antioxidant activity, total flavonoids and phenolic content, radio-protective potential in correlation to the prevention of protein oxidation and DNA strand breaks in vitro. The carotenoid pigment showed a very potent antioxidant activity and significantly stronger scavenging ability against superoxides, with an IC50 (concentration causing 50% inhibition of the desired activity) of 41.6 μg/ml. The total phenolic and flavonoid contents were 12.1 and 7.4 μg in terms of gallic acid and quercetin equivalents per milligram of dried mass, respectively. Astaxanthin also showed a higher inhibitory action against oxidative damage to collagen, elastin and bovine serum albumin than did β-carotene. The carotenoid also inhibited breaks to DNA strands, as indicated by the results of the DNA damage prevention assay. We conclude that astaxanthin from Deinococcus sp. strain WMA-LM9 has protective effects against radiation-mediated cell damage, and it also protects cellular protein and DNA against oxidative stress and other anti-oxidant activities.

Keywords: Deinococcus sp., radio-resistance, astaxanthin, HPLC, NMR, MS, antioxidant, protein oxidation,

5.2. Introduction Extremophiles are all those organisms that can survive and optimally grow in extreme conditions e.g. volcanic areas, deep seas, hot springs, as well as in environments with oxygen shortage (Kumar et al., 2010). Among these extremophiles, radio-resistant are very important because they can survive under high radiations (both ionizing & non-ionizing). These radiations have severe effects like oxidative damage to biomolecules such as nucleic acid and proteins. Extreme energy radiations tolerance has been detected in various members of archaea and bacteria domains. Most important genera having ionizing-radiation-resistance includes

Page 179

Rubrobacter and Deinococcus that exhibits a high level of resistance (Fredrickson et al., 2004). Some other extremely UVC radiations resistant bacteria have been reported from the Atacama Desert Chile, Sonoran Desert Arizona and from manganese mine in northern Argentina that includes Hymenobacter sp. and Geodermatophilus sp. The isolates comprised 28 genera grouped within six phyla, which we ranked according to their resistance to UVC. Hymenobacter sp. showed a higher survival profile than D. radiodurans a bacterium, generally considered the most radiation-resistant organism (Rothschild et al., 2016). Many radiation-resistant organisms with desiccation resistance have been reported from dry areas in Xinjiang, Taklimakan Desert and displayed diverse metabolic properties. A total of 52 Ɣ-radiation-resistant bacteria were isolated from the Taklimakan desert sample that were clustered into five group’s based on the 16S rRNA sequencing (Yu et al., 2015). These extremophiles (radio resistance) protects themselves by synthesizing certain antioxidants to hostage oxygen radicals or else they have a well-developed DNA repair mechanisms (Betlem et al., 2012). Further down penetrating light, bacteria crease excess light and quench what is not required, thus escaping undesirable photochemical impairment. Deinococcus radiodurans is one of the most studied radio-resistant, red pigmented and nonphotosynthetic bacteria. The red pigment is assumed for the microbial resistance against highly energetic radiation (Battista and Cox, 2005).

Carotenoids have no role in normal cell growth, reproduction or development of organisms, but its absence does upset the survivability. These are effectual trackers of reactive oxygen species 1 (ROS), predominantly that of singlet oxygen O2 and peroxyl radicals (ROO) (Tatsuzawa et al., 2000; Stahl and Sies, 2003). These metabolic assets of microbes have widely been studied for various industrial uses (Ferrer et al., 2007; Gostincar et al., 2010). The assembly of carotenoids from natural sources has been a region of demanding exploration (Scarpa et al., 2015). D. radiodurans produces deinoxanthin that is a unique keto carotenoid, which contributes to D. radiodurans resistance under oxidative stress with a potent quenching ability of ROS than - carotene and lycopene (Saito et al., 1998; Tian et al., 2007; Peng et al, 2009). In fact, carotenoids defend DNA strands from oxidative damage, membranes from lipid peroxidation and proteins from carbonylation (Zhang and Omaye, 2000).

− All types of ROS like hydroxyl radicals (•OH), hydrogen peroxide (H2O2), superoxide (O2 ), and 1 singlet oxygen ( O2) and reactive nitrogen species (RNS) like 2, 2-diphenyl-1-picrylhydrazyl (DPPH) can be hunted by carotenoids from D. radiodurans in vitro (Tian et al., 2007; Zhang et al., 2007). These carotenoids also have the ability to protect plasmid DNA (20%) uncover to OH, and recovers the supercoiled plasmid form, that is completely crushed (Tian et al., 2009). The advancement in metabolomics, proteomics, and genomics, has increased the interest to study the genes and its proteins that are helpful in regulation of microbial metabolic assets in such an extreme environment (Ferrer et al., 2007; Hammon et al., 2009; Singh et al., 2010).

Page 180

Figure 5.1: Astaxanthin synthesis from isophorone, cis-3-methyl-2-penten-4-yn-1-ol and a symmetrical C10-dialdehyde has been discovered and is used commercially.

Figure 5.2: Structure of Deinoxanthin from Deinococcus radiodurans.

Accordingly, in current study we investigated the role of astaxanthin extracted from

Deinococcus sp. strain WMA-LM9 in a resistance to UVB, H2O2 and Mitomycin C. The carotenoid was purified and investigated for antioxidant and cytotoxic activity. Oxidative damage in bovine serum albumin, collagen and elastin with and without carotenoids and their possible role in Deinococcus sp. WMA-LM9 resistance to intracellular protein carbonylation was also studied. We also confirmed that these carotenoids can neutralize the effect of different reactive oxygen species resulting from oxidative stress that can damage microbial DNA. To the best of our knowledge, this is the first report of astaxanthin-producing Deinococcus with highly anti-oxidant and protein oxidation preventing activities.

Phenolic Flavonoid’s

Figure 5.3: Structure of phenolics and flavonoids

Page 181

5.3. Material and methods 5.3.1. Isolation of Radio-resistant Bacteria Soil samples were collected aseptically from the desert of District Lakki Marwat, Khyber Pukhtoonkhwa, Pakistan, in sterile zipper plastic bags, immediately transported to the laboratory and kept at 4°C before investigation for the radioresistant microbial community. Soil samples were serially diluted in phosphate buffer saline (PBS) and inoculated by spread plate method on TGY (Trypton glucose yeast extract agar) medium containing 10 g/L tryptone, 5 g/L YE, 1 g/L glucose, was used as basic medium. TGY plates were exposed to UV radiation for 5 min prior to incubation. Each sample was UV irradiated in UV chamber (119x55×52 cm), which was supplied with a 20W, 280 nm UV light placed at the top. The choice of UV-B was based on the fact that UV 280 nm can cause a serious damage to cell DNA because of its shorter wavelength. Moreover UVB is considered to be the only choice for causing both types of direct and indirect damages.

The UV fluence rate (energy per area per time) to the test sample was calculated by using the following equation in J/m2.

Where He is the radiant exposure that is the energy reaches a surface area due to irradiance (Ee) maintained for a time duration (t).

5.3.2. Radiant Exposure Calculation TGY plates were exposed to UV radiation for 5 min prior to incubation. Each sample was UV irradiated in UV chamber (119x55×52 cm), which was supplied with a 20W, 280 nm UV light placed at the top.

Total UV dose was determined by time of exposure to the UV fluence rate. All UV irradiation procedures were performed under red light to prevent photoreactivation. After irradiation, the plates were incubated at 37ᵒC for 5-7 days. The isolates were subcultured from irradiated plates and again exposed to UV radiation for further confirmation. Several fractionated doses ranging from 300 to 3300 Jm-2 (30 to

300 sec) were used to find the survivability rate of all the UVR isolates. Strain WMALM9 was selected on the basis of tolerance to maximum doses of UV radiation.

5.3.3. Identification of Radio-resistant Bacterium

Based on high tolerance to UV radiations, H2O2 and mitomycin C strain WMA-LM9 was identified morphologically as well as biochemically by previously described methods (Shah et al., 2013). Molecular identification was carried out by sequencing of 16S rRNA gene. For this purpose, for the extraction of DNA extraction kit (qiagen) was used. Amplification of 16S rRNA gene was carried out by 27F’ (5’-

AGAGTTTGATCCTGGCTCAG-3’) and 1492R’ (5’- CTACGGCTACCTTGTTACGA-3’) bacterial primers.

The amplified PCR product was sequenced by Macrogen Service Center (Geunchun-gu, Seoul, South Korea). The obtained sequence was computed for closest relatives using BLAST tool

Page 182

at the NCBI and homologues were analyzed for phylogeny using Molecular Evolutionary Genetic Analysis (MEGA) version 6. The neighbour-joining phylogenetic (Tamura and Nei, 1993) tree was constructed for the identification of isolated bacterial strain and diversity among UVR resistant extremophiles was studied (Tamura et al., 2013). Afterwards, the sequence was submitted to NCBI GeneBank in order to assign an accession number (KT008384).

5.3.4. Bacterial Survival Curves and Oxidative Stress Strain WMA-LM9 was inquested for radiation resistance and survival curve was plotted (Mattimore and Battista, 1996). Cell culture of strain WMA-LM9 was serially diluted (1:1000) by phosphate buffer saline (PBS) and spread on TYG agar plates, then exposed to different doses of UV radiation at 280 nm. For the determination of survival rate UV-irradiated plates were divided by colonies from unirradiated plates. For determination of oxidative stress and mitomycin C tolerance, an overnight grown culture of strain WMA-LM9 in TGY broth was diluted in sterile normal saline up to an OD600 of 0.5. The cells suspension was treated with different molar concentrations of hydrogen per-oxide (5-40 mM) for 30 min and mitomycin C (2-10 µg/ml) for 20 min, cell were cultured on TGY agar plates. Former to persisting colonies counting, plates were incubated at 30°C for 3 days. The survival rate was expressed as the difference in number of colonies between treated and untreated samples. All the experiments for survival curve were run in triplicates.

5.3.5. Measurement of Intracellular Protein Carbonylation Protein carbonylation was measured using the DNPH (2,4-dinitrophenyl hydrazine) method (Cao and Cutler, 1995; Misra et al., 2004). To obtain cell free extract for protein carbonylation assay cells were lysed by sonication. For the estimation of total protein concentration method by Lowry et al., (1951) was used. The cell-free extract (2 mg/ml of protein) in 50 mM PBS (pH 7.4) was incubated with 400 µl of 10 mM 2,4-dinitrophenyl hydrazine (DNPH) in 2M HCl for 2 hrs. Proteins were precipitated, and then re-suspended in 6M guanidine hydrochloride. After centrifugation of solution supernatant was analysed spectrophotometrically at 370 nm. As protein control DNPH was replaced with 2M HCI, was run in parallel. The protein carbonyl content was expressed in mM/mg protein.

5.3.6. Preparation of the Carotenoid Extract 500ml of the Deinococcus sp. WMA-LM9 culture under continuous shaking and aerobic conditions was harvested after 48 h by centrifugation for 10 min at 5000xg.Cell pellet was extracted with [acetone:methanol:chloroform (7:5:3)], after washing with sterilized water by probe sonication (150 watts of power at 40 kHz). The cell suspension was again centrifuged at 10000 × g for 10 min and a clear red colour supernatant was recovered. It was allowed to dry and then dissolved in methanol for further study.

5.3.7. Reversed-Phase High-Performance Liquid Chromatography (R-P HPLC) The crude extracts were inspected by HPLC and flash chromatography using a Waters 2690 Alliance system. A Hypersil ODS-C18 column (5 µm pore size, 4.6 × 250 mm) protected by a

Page 183

guard column. All solvents of HPLC grade were degassed (Merck, Millipore Corporation, Merck KGaA, Darmstadt, Germany) and filtered through a 0.2 µm filter prior to HPLC analysis. Using standard column thermostat, constant temperature of 25°C was maintained in column. An isocratic technique eluted with a mixture of isopropanol, methanol and acetonitrile (10:50:40, v/v) at a flow rate of 0.8 ml/min was used as mobile phase (Saito et al., 1998). The fractions eluted were monitored with a Waters 996 photodiode array detector.

5.3.8. Liquid Chromatography–tandem Mass Spectrometry Analysis The carotenoid extracts from Deinococcus sp. WMA-LM9 was subjected to LC-MS/MS an ABX3200 Q-TRAP mass spectrometer equipped with a

TurbolonSpray ESI source, and connected to a Shimadzu HPLC system with dual LC20 pumps, a SPD-M20A UV/Vis photodiode array (PDA) detector and auto sampler. 10 μL of the sample dissolved in LC-MS grade methanol were injected onto a column

(C18; 5 μm, 250 × 4mm, Bischoff, Germany). The mobile phase comprised of Methanol (solvent A) and Acetonitrile (solvent B) with 0.1% (v/v) formic acid, used in a gradient mode for B: 0.0/30; 25/100; 35/100; 45/30 (min/%); with a flow rate of 0.8ml/min. A computer equipped with Thermos Scientific Xcalibur 2.2 (Thermo Fisher Scientific, USA) was used to analyze data in control manner. The system was controlled and data were analyzed on a computer equipped with Thermo Scientific Xcalibur 2.2 (Thermo Fisher Scientific, USA). The MS was used in positive ion mode to detect m/z transitions [M+H]+.

5.3.9. 1H and 13C NMR Studies A nuclear magnetic resonance spectrum gives the prime evidence about the structure of a compound. NMR spectroscopic data were recorded at room temperature on Bruker Avance 400

MHz NMR spectrometer in CDCl3 referenced to residual

H C 77.0 ppm) with tetramethylsilane (TMS) as an internal standard.

The purified sample was placed in an inert solvent [deuteron-chloroform (CDCl3), and the solution was positioned between the poles of a powerful magnet. In respond to their molecular environs the diverse chemical shifts of proton and carbon within molecule were measured in the NMR apparatus relative to a standard, usually tetramethylsilane (TMS). The strength of the signals was combined to reveal the number of carbons and protons resonating at any one frequency. Each chemical shift value corresponds to a set of protons and carbons in a particular environment. The strength of each signal signifies the number of protons and carbon of each type.

5.3.10. Anti-Oxidant Activity and Determination of Total Phenolic/Flavonoid Contents

To determine the anti-oxidant activity of carotenoid Commonly used Diphenyl-1- Picrylhydrazyl (DPPH) assay was used (Xu et al., 2005). Various concentrations of carotenoid (5-20 µg) were taken in 96 well microtiter plate, volume was raised uniformly up to 200 µl using DPPH and incubated for 30min at 37°C. Ascorbic acid was taken as a standard in same concentration to the test samples. Methanol was used as a blank and absorbance was measured at 517 nm on UV-

Page 184

visible spectrometer. The IC50 values were calculated for carotenoid along with standard. The following formula was used for calculation of free radical scavenging activity.

% Scavenging = (Control-Sample/Control) x 100

Total phenolic and flavonoid phenolic contents were analyzed in the extracted carotenoid from Deinococcus sp. strain WMA-LM9. Singleton et al (1999) method was used for the determination of total phenolic content (TPC) using gallic acid as standard. While total flavonoid contents were calculated by aluminium chloride colorimetric assay (Zhishen et al., 1999) by plotting quercetin standard curve.

Phenolic and flavonoid contents were measured in µg of gallic acid and quercetin equivalents per milligram (GAE/mg and QE/mg) of dried extract.

5.3.11. Protein Oxidation Inhibition Assay Elastin, collagen and bovine serum albumin as standard proteins were used to analyze inhibitory effect of carotenoid on protein oxidation. Two hundred micro-litter of targeted proteins (1mg/ml) were incubated with hundred micro-litter of carotenoid dissolved in tetrahydrofuran

(THF) and treated with hundred micro-litter of (1 mmol/L) FeSO4 and 100 µl of 80 mmol/L of H2O2 at 37oC for 1 hr. 15 U of catalase was added to stop the reaction. The mixture was then incubated with 600 µl of 10 mmol/L DNPH for 1 hr. 10% TCA was added to precipitate out the unbound protein afterwards. The mixture was dissolved in 6M guanidine hydrochloride and quantified spectrometrically at 370 nm. Percent inhibition of protein oxidation by carotenoids was calculated using H2O to replace FeSO4 and H2O2 as a blank.

(%) Inhibition of protein oxidation= (Control-sample/control) x 100

5.3.12. DNA Damage Prevention Assay The DNA damage preventing capability of carotenoid pigment was determined by incubating plasmid pUC18 in a reaction mixture that contains: plasmid 2 µl, carotenoid solution 3

µl (6 µg) and 6 µl (12 µg), 2mM FeSO4 3 µl, 1M sodium nitroprusside 4 µl and 30% H2O2 4 µl, for 1hr at room temperature. Hydrogen per oxide and sodium nitroprusside usually produces single strand breaks in DNA. The pattern of bands of treated samples, as well as positive and negative control, was examined using gel electrophoresis technique.

5.3.13. Cytotoxic Assay Brine shrimp assay was performed to analyze cytotoxicity of the carotenoid (Maridass, 2008). In distilled water 34 gm of sea salt per litre was added for the prepration of artificial seawater and taken in vials with 15-20mg eggs of brine shrimp (Artemia salina). Carotenoid dissolved in DMSO, was added to vials in different volume (100µl, 50µ, 25µl) and incubated for 24-48h at 30°C. Cytotoxicity of carotenoid was determined by counting the number of live shrimps.

5.3.14. Statistical Analysis

To assess the significance between the results, Student’s t-test was used and P < 0.05 was considered as significant. Bacterial sensitivities to UV, H2O2 and mitomycin C and scavenging %

Page 185

activity of carotenoid were studied by regression analysis between the percentage of survival/inhibition and their respective concentrations, single factor and two-way ANOVA applied for analysis of protein carbonylation and in vitro % protein oxidation inhibition assay between groups and within single group.

5.4. Results 5.4.1. UVB Selection and Isolation of Radio-resistant Bacteria The shorter wavelength UVB (280 nm) radiations carry 3.94–4.43 eV energy per photon and can damage cellular DNA.most common types of DNA damage are pyrimidine-pyrimidone (6- 4) photoproducts (6-4PPs) and cyclobutane pyrimidine dimers (CPDs) that can be caused by UVB and lead to CC-TT or C-T transitions.

5.4.2. Identification of Strain WMA-LM9 Strain WMA-LM9 was found Gram-positive diplococcus, tetrad in arrangement, with round, red, raised mucoid and opaque colonies. 16S rRNA sequence of strain WMA-LM9 indicated that strain belongs to genus Deinococcus having 99% similarity with Deinococcus radiopugnans. The strain was submitted to NCBI under accession number KT008384.

5.4.3. Resistance to UV radiation, Oxidative Stress and Mitomycin C Strain WMA-LM9 was exposed to different energy doses of UV radiation in order to determine its radiation resistant potential. Strain WMA-LM9 maintained nearly 50% viability at 5K Jm-2 energy dose of UV radiation, whereas the E. coli (10536) couldn't survive at such high energy UV radiation (Fig. 5.4A). A gradual decrease in survival of strain WMA-LM9 was observed with increase in concentration of H2O2 and maintained up to 49% viability at 50mM H2O2 for 60min (Fig. 5.4B). The bacterium was quite resistant to mitomycin C and more than 50% of survival rate was observed up to 8µg/ml whereas the E. coli couldn't even survive at this concentration (Fig. 5.4C). Results are expressed as means ± SD and are compared using the Student’s unpaired t-test. Moreover, the percentage values had an exponential distribution. Error bars represent standard deviation for triplicate experiments. P value < 0.05 is considered significant.

5.4.4. Measurement of Intracellular Protein Carbonylation Level The total protein oxidation in Deinococcus sp. WMA-LM9 and E. coli (10536) was measured as 0.128091± 0.00585 and 0.197378 ± 0.0191 µM/mg respectively that indicates lower protein oxidation in the radio-resistant WMA-LM9 sp. as compared to E. coli (10536). The results shown in figure 3 indicated that lack of ability to produce carotenoids in E. coli (10536) becomes more sensitive to oxidative damages like UV stress and H2O2 (Fig. 5.5). Results are highly significant and expressed as means ± SD with p<0.05.

Page 186

Figure 5.4: Survivability of strain WMA-LM9 from desert soil at varying UV-B exposure. (A) UV radiation resistant potential of WMA-LM9 (B) Resistance to different concentrations of hydrogen peroxide (mM) (C) Resistance to different concentrations of Mitomycin C (µg/ml). % viability value is calculated as N1/N0x100 where Ni is the value after exposure to irradiation, H2O2 and Mitomycin C, while N0 is the value at time 0, for each condition tested. Results are highly significant different in among group (Fig. 5.4) as p value is less than significant level 0.05. Values are mean ± SD.

5.4.5. Carotenoid extraction and purification The carotenoids were extracted from the cells of Deinococcus sp. WM-LM9 and analyzed by HPLC. The fraction 7 of flash chromatography (Fig 5.6) showing highest scavenging activity was subjected to RP-HPLC. Two distinct peaks were observed with a retention time of 2.3 and 4.9

Page 187

collected separately marked as LM9F1 LM9F2 as shown in Fig. 5.7. 15 mg of pure astaxanthin was extracted from Deinococcus sp. WM-LM9 with percent purity of more than 90.

5.4.6. LCMS/MS The fraction LM9F1 that exhibited the highest antioxidant activity was subsequently subjected to LC–MS/MS for mass identification for C40H52O3 by elemental analysis and electron ionization liquid chromatography (EIMS). The positive ESI-MS spectrum of carotenoid extract exhibits the signals at m/z 597 [M+H] + (LM9F1) and 610 [M+H] + (LM9F2) (Fig. 5.7). The major component, m/z 596 accounted for more than 70% of the total carotenoids. The UV spectrum of the compound showed λmax at 475 nm.

5.4.7. 1H and 13C NMR

The purified extract once dissolved in an inert solvent [deuteron-chloroform (CDCl3) was analysed for carbon and proton spectra to investigate the chemical structure properties. The peaks obtained by NMR spectroscopy were found to contain astaxanthin. All the peaks were compared with available spectra of astaxanthin and confirmed. double bonds. The formation of cis bond results in characteristic shift differences compared to all- trans compounds. The proton NMR data shows that signals between 6.0 and 7.8 ppm represent the 14 (-CH) methine protons on the ASTX backbone and a near bilateral symmetry around the central double bond. The conjugation system described imparts carotenoids with excellent light absorbing properties in the bluegreen (450–550 nm) range of the visible spectrum. The signals in range of 0.99 to 2.1158 ppm represents 30 protons from methyl group and two OH protons show peak at 3.627ppm. In addition, the presence of unsaturated fatty acids is detectable by the appearance of multiplets between 5.2–5.4 ppm attributed to methine protons (Fig. 5.8).

In the 13C NMR spectra, the signals attributed to the carbon atoms found in the carbonyl moieties are good indicators of structure of astaxanthin. The C-1 is a quaternary carbon and it gives peak at 29.69ppm. In the spectrum C-4 (C=O) signal is downfield at 200.14 ppm. The 13C NMR spectra of compound reveals the presence of C=C carbon-3 position with 162.27 ppm on both side of ring showing the presence ester carbonyl. It also confirms the monoester linkage in the compound. C- 5 an alpha carbon to carbonyl and olefinic region shows peak at 126.76-136 ppm. The 8 methyl groups at these olefinic carbons show peaks at 14.00 and 26.13 ppm (Fig. 5.9). The signals corresponds to the carbon atoms of the carotenoid were found to be the same as those of already reported astaxanthin (Fig. 5.10) in literature.

1 H NMR spectroscopy of the pure extract showed diastereomeric arrangement in the olefinic region that gives a significant evidence for the presence of carbohydrate backbone in carotenoids. NMR spectra showed hydrophobic nature of the pure compound arranged in isoprene residues with long conjugated chain of double bonds. Therefore, our attention was directed to the chemical shift area for

Page 188

Figure 5.5: Comparison of intracellular protein carbonylation level between radioresistant

Deinococcus sp. strain WMA-LM9 and E.coli (10536) following UVR and H2O2 treatment. Results are highly significant different with p value less than 0.05. Values are mean ± SD.

Page 189

Figure 5.6A: Flash chromatography of the extract using different solvent system of Hexane Dichloromethane water and methanol.

Figure 5.6B: Different fractions collected upon flash chromatography for bioassay guided fractionation.

Page 190

Figure 5.7: HPLC chromatogram/positive ESI-MS spectrum of carotenoid extract exhibits signals at m/z 597 [M+H]+, and 609 [M+H]+ LM9F1 and F2 respectively.

Figure 5.8: 1H NMR spectra of purified compound LM9F1. The purified compound was identified as ―Astaxanthin‖

Page 191

Figure 5.9: 13C NMR spectra of purified compound LM9F1. The purified compound was identified as ―Astaxanthin‖

Figure 5.10: Astaxanthin chemical structure from NMR peaks using ChemDraw software. 5.4.8. Anti-oxidant Activity of Carotenoid Methanol dissolved carotenoid extract DPPH· radical scavenging activity was measured as shown in figure 5.11. Concentration dependent scavenging activity was found to and almost 50%

(IC50) of the DPPH· radicals were scavenged at 41.6 µg/ml carotenoid that was greater that the activity of -carotene (20%). However, the carotenoid extract scavenged less DPPH radical (80 ±

1.7%) than ascorbic acid positive control. IC50 was obtained by linear regression analysis of dose response curve plotting between % inhibition and carotenoid concentration that led to 50% inhibition of free radical activity of DPPH. Results are expressed as means ± SD or SE and are compared using the Student’s unpaired t-test p<0.05.

Page 192

The phenolic and flavonoid contents in the carotenoid extract (astaxanthin) were quantified by using standard calibration curve equation. Various concentrations (5-20 µg) of gallic acid and quercetin were used as standards to plot calibration curve and the results were expressed as µg of standard equivalents. Total phenolic and flavonoid contents were 12.1 ± 1.3 and 7.4 ± 1.0 µg/mg of the respective standards equivalence (µg of GAE/mg and µg of QE/mg) from the calibration curve equation.

5.4.9. Protein Oxidation Inhibition Assay The inhibitory effect of astaxanthin from strain WMA-LM9 against oxidative damage of three standard proteins i.e., bovine serum albumin, collagen and elastin were studied, using - carotene as standard. Astaxanthin is able to inhibit protein oxidation by 40-45% ± 10.3, which is a greater inhibition than that achieved with carotene (15-20 ± 3.3%). 10 µg of this carotenoid inhibited protein oxidation better than -carotene, with significant results as p<0.05 using t-test as shown in Fig. 5.12.

5.4.10. Inhibition of Protein Oxidation and DNA Damage Prevention The effect of astaxanthin on DNA damage prevention was examined using a hydroxyl radical-induced DNA breaks system in vitro. Specifically, plasmid pUC18 was incubated with H2O2 and sodium nitroprusside in the presence and absence of carotenoid. The plasmid DNA was broken down by the attack of the •OH generated from the Fenton reaction, as indicated by smear formation in the negative control in Fig. 5.13. The DNA was completely protected from oxidative damage by H2O2 and sodium nitroprusside by the presence of carotenoid in test samples T1 and T2 (6 and

12 μg) in the reaction mixture, showing promising results in DNA prevention (Fig 5.13).

5.4.11. Cytotoxic Activity of Carotenoid Cytotoxic activity of astaxanthin was determined by brine shrimp assay. For the evaluation of cytotoxic effect assay was carried out at four different concentrations of carotenoid. At lowest concentration of carotenoid no toxic effect was noticed that is 25-100 µg, while at higher concentrationts only 30% cytotoxicity was observed with an IC50 of 1567.62 µg.

Page 193

Figure 5.11: Anti-oxidant activity of carotenoid extracted from strain WMA-LM9. DPPH radical 50% (IC50) was obtained by linear regression analysis of dose response curve plotting between % inhibition or % activity (y-axis) and carotenoid concentration (41.6 µg/ml).

Page 194

Figure 5.12: Inhibitory effect of carotenoid from strain WMA-LM9 of different protein oxidation in-vitro. Commercially available β-carotene (Sigma Aldrich) was used as standard. Values are mean ± SD.

Figure 5.13: Role of carotenoids in prevention of oxidative damage to pUC18 plasmid DNA after exposure to oxidative agents. Lane PC: Positive control (only plasmid DNA); Lane NC: Negative control (plasmid DNA treated with hydrogen peroxide and sodium nitroprusside). Lane T1 and T2: Test samples (plasmid DNA, hydrogen peroxide, sodium nitroprusside and carotenoid in different concentration).

Page 195

5.5. Discussion Hot and dry desert can be considered as paradigm of extreme environment for all forms of life because of several limiting factors i.e. nutrient availability, extreme dryness, and high temperature. Continuous exposure to high sunrays and dryness, it may receive an ample amount of radiations. In current years, mechanism underlining ROS-induced oxidative stress mechanisms and the exploration for appropriate strategies to fight oxidative stress has become one the foremost goal of medical research efforts (Vílchez et al., 2011). In the present study surface soil, collected from the desert of District Lakki Marwat, was screened for the isolation of radio- resistant bacteria. Carotenoid extract from this bacterium was purified and evaluated for DPPH radical, protein and DNA oxidation inhibition activity.

A radio-resistant bacterium, Deinococcus sp. strain WMA-LM9 was isolated from desert soil. Strain WMA-LM9 showed prolonged resistance (50% survivability) to different energy doses of UVB radiation and also was found viable after incubating with mitomycin C (10 µg/ml) for 20 min, 50mM hydrogen peroxide for 60 min suggested that this strain has a strong CAT (catalase) and SOD (super oxide dismutase) antioxidant system that protects the cell from oxidative damages (Prazdnova et al., 2014). It has already been reported by several researchers that resistance to ionizing radiation is directly linked with resistance to hydrogen peroxide, mitomycin C and desiccation (Daly et al., 2007; Fredrickson et al., 2008; Daly 2009).

Deinococcus strain’s resistance to high concentration of mitomycin C for 10 min generates 100 to 200 cross-links per genome without a loss of viability (Kitayama,i 1982). However, the detailed antioxidant mechanisms of this bacterium are still unknown.

The carotenoid compound in LM9F1 was analysed for astaxanthin (C40H54O4) by elemental analysis and EIMS giving molecular ion at 597 m/z. Astaxanthin protected cellular proteins from oxidative damage in case of strain WMA-LM9 as compared to E. coli (10536). The carotenoids are effective scavengers of different toxic oxides, therefore, block the formation of all superoxide’s and Fenton reaction pathways that can contribute to protein oxidation (Imlay, 2003; Hua et al., 2009). The ability of this genus to survive in several extreme conditions is suggested to be as a result of three combined mechanisms like prevention, tolerance and repair (White et al., 1999).

The mono-esterified astaxanthin from WMA-LM9 showed two-fold stronger quenching abilities of super oxides than already reported deinoxanthin and carotenes, that might be attributed to the extra keto-group substitution and length of their conjugated double bond system as compared with the other carotenoids. Antioxidant potential of carotenoids from radio-resistant microbes has been documented as the contributory factor to radioprotection presented by any compound (Albrecht et al., 2000). A carotenoid extract from D. radiodurans was clearly able to scavenge superoxide anions using the DPPH assay (Zhang et al., 2007).

Free radicals formed during DPPH assay showed that astaxanthin have capability of donating electrons to neutralize free radicals and can scavenge free radicals and therefore has potential as chemotherapeutic drugs to eradicate pathological diseases related to free radical from a system. The photo-protection against toxic super-oxides offered by astaxanthin is based on electron exchange energy transfer quenching (Galano et al., 2010). Protein rather than DNA was proposed to be a possible target for UVB radiations and free radicals. The carotenoid isolated from this bacterium was more effective to prevent the oxidation of different standard proteins like bovine serum albumin, collagen and elastin. The protective effect of purified astaxanthin on

Page 196

proteins in Deinococcus sp. is the result of free radical quenching ability. DNA repair proteins and many other important cell enzymes and proteins involve in cell recovery are protected by these naturally occurring carotenoids in the cells.

Carotenoid form stable product upon reaction with free radicals and additional reactions at their conjugation double bonds, which further contribute to the inhibition of protein and lipid damages from oxidative products formed during stress (Krinsky et al, 2005; Hua et al., 2014). We also investigated that this newly reported monoesterified astaxanthin from WMA-LM9 neutralized the effect of superoxide’s, hydrogen peroxide and sodium nitroprusside and prevented pUC18 plasmid DNA from oxidative damage. It also restricted protein oxidation by inhibiting protein carbonylation that led to prevention of DNA indirect damage. The carotenoid might block the formation of 8-oxo-2-deoxyguanosine during oxidation of DNA in stress. Many important proteins including DNA repair proteins and other enzymatic antioxidant are protected by carotenoids in order to prolong cells survivability in extreme conditions. Antioxidant-rich metabolites from radio-resistant extremophiles significantly reduce the risk of DNA damages (Singh and Gabani, 2011).

The carotenoid was found either non-toxic or less toxic even with higher concentration that makes it more effective to use in drugs and other therapeutic applications. Little is known about the total phenolic and flavonoid contents in carotenoid of Deinococcus radiopugnans, which may contribute to the radioprotective ability and anti-oxidant activity of these compounds. As the presence of aromatic hydro carbons, double bonding system and different keto groups indicate the high resonance structure of the extracted carotenoid from Deinococcus sp.WMALM9. So we investigate the phenolic and flavonoid groups that can contribute to antioxidant and DNA damaging preventive abilities for such carotenoids. Mostly flavonoids imparts a characteristic colors (orange, violet, crimson, scarlet, and mauve, blue) with beneficial health effects ( ili et al.,

2012). The OCH3 groups shifts the color toward more red (Grotewold 2006). The presence of oxo groups at position 4 and nine or more double bonds in the carotenoids enhances singlet oxygen and super oxides quenching activities (Terao, 1989). Contribution of the carotenoids and phenolic contents to the radical scavenging activity was described by several researches previously (Fernandez et al., 2007; Wang et al., 2010). Our results confirm that both carotenoids and phenolic are contributing to the radical scavenging property of the extract. Carotenoid with high phenolic and flavonoid spectrum determines its medicinal importance. Deinococcus sp. strain WMA-LM9 has higher levels of CAT, hence more resistant to UV, mitomycin C, and hydrogen peroxide than Escherichia coli.

The results concluded that astaxanthin from the newly isolated Deinococcus sp. WMA- LM9 play a key role in protection against UV-photo-oxidation, protect proteins and DNA from oxidative damage and contribute to cell resistance. Furthermore investigations are required to find its biosynthetic pathway in order to produce highly active carotenoid via metabolic engineering.

Page 197

References

1. Albrecht, M., Takaichi, S., Steiger, S., Wang, Z.Y. and Sandmann, G., 2000. Novel hydroxycarotenoids with improved antioxidative properties produced by gene combination in Escherichia coli. Nature biotechnology, 18(8), pp.843-846. 2. Betlem, P., Bovens, P., Dings, T., Vriend, S., 2012. Radioresistance: A Search for Carotenoids in Rubrobacter radiotolerans. Radboud Honours Academy FNWI-Radboud University Nijmegen. In Dei Nomine Feliciter 1– 51. 3. Cao, G.H. and Cutler, R.G., 1995. Protein oxidation and aging 1. Difficulties in measuring reactive protein carbonyls in tissues using 2, 4dinitrophenylhydrazine. Archives of biochemistry and biophysics, 320(1), pp.106-114. 4. Cheng, J., Zhang, Z., Zheng, Z., Lv, G., Wang, L., Tian, B. and Hua, Y., 2014. Antioxidative and Hepatoprotective Activities of Deinoxanthin-Rich Extract from Deinococcus radiodurans R1 against Carbon TetrachlorideInduced Liver Injury in Mice. Tropical Journal of Pharmaceutical Research, 13(4), pp.581-586.

5. Cox, M.M. and Battista, J.R., 2005. Deinococcus radiodurans—the consummate survivor. Nature Reviews Microbiology, 3(11), pp.882-892. 6. Daly, M.J., 2009. A new perspective on radiation resistance based on Deinococcus radiodurans. Nature Reviews Microbiology, 7(3), pp.237-245. 7. Daly, M.J., Gaidamakova, E.K., Matrosova, V.Y., Vasilenko, A., Zhai, M., Leapman, R.D., Lai, B., Ravel, B., Li, S.M.W., Kemner, K.M. and Fredrickson, J.K., 2007. Protein oxidation implicated as the primary determinant of bacterial radioresistance. PLoS biology, 5(4), p.e92. 8. Edge, R., McGarvey, D.J. and Truscott, T.G., 1997. The carotenoids as anti-oxidants—a review. Journal of Photochemistry and Photobiology B: Biology, 41(3), pp.189-200. 9. Fernandez, E., Galvan, A., 2007. Inorganic nitrogen assimilation in Chlamydomonas. Journal of Experimental Biology 58:2279–2287. 10. Ferrer, M.G., Beloqui, O. and Golyshin, A., PN (2007). Mining enzymes from extreme environments. Curr Opin Microbiol, 10, pp.207-214. 11. Fredrickson, J.K., Shu-mei, W.L., Gaidamakova, E.K., Matrosova, V.Y., Zhai, M., Sulloway, H.M., Scholten, J.C., Brown, M.G., Balkwill, D.L. and Daly, M.J., 2008. Protein oxidation: key to bacterial desiccation resistance?. The ISME journal, 2(4), pp.393- 403.

Page 198

12. Fredrickson, J.K., Zachara, J.M., Balkwill, D.L., Kennedy, D., Shu-mei,

W.L., Kostandarithes, H.M., Daly, M.J., Romine, M.F. and Brockman, F.J., 2004. Geomicrobiology of high-level nuclear waste-contaminated vadose sediments at the Hanford Site, Washington State. Applied and environmental microbiology, 70(7), pp.4230- 4241.

13. Galano, A., Vargas, R. and Martínez, A., 2010. Carotenoids can act as antioxidants by oxidizing the superoxide radical anion. Physical Chemistry Chemical Physics, 12(1), pp.193- 200. 14. Gostinčar, C., Grube, M., De Hoog, S., Zalar, P. and Gunde-Cimerman, N., 2009. Extremotolerance in fungi: evolution on the edge. FEMS microbiology ecology, 71(1), pp.2- 11. 15. Grotewold, E., 2006. The genetics and biochemistry of floral pigments. Annu. Rev. Plant Biol., 57, pp.761-780. 16. Hammon, J., Palanivelu, D.V., Chen, J., Patel, C. and Minor, D.L., 2009. A green fluorescent protein screen for identification of well expressed membrane proteins from a cohort of extremophilic organisms. Protein Science, 18(1), pp.121-133. 17. Imlay, J.A., 2003. Pathways of oxidative damage. Annul Review Microbiology 57:395–418. 18. Kitayama, S., 1982. Adaptive repair of cross-links in DNA of Micrococcus radiodurans. Biochimica et Biophysica Acta (BBA)-Gene Structure and Expression, 697(3), pp.381-384. 19. Krinsky, N.I. and Johnson, E.J., 2005. Carotenoid actions and their relation to health and disease. Molecular aspects of medicine, 26(6), pp.459-516. 20. Kumar, R., Patel, D.D., Bansal, D.D., Mishra, S., Mohammed, A., Arora,

R., Sharma, A., Sharma, R.K. and Tripathi, R.P., 2010. Extremophiles: sustainable resource of natural compounds-extremolytes. In Sustainable Biotechnology (pp. 279-294). Springer Netherlands. 21. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J biol Chem, 193(1), pp.265-275.

Page 199

22. Maridass, M., 2008. Evaluation of brine shrimp lethality of Cinnamomum Species. Ethnobotanical Leaflets, 2008(1), p.106. 23. Mattimore, V. and Battista, J.R., 1996. Radioresistance of Deinococcus radiodurans: functions necessary to survive ionizing radiation are also necessary to survive prolonged desiccation. Journal of bacteriology, 178(3), pp.633-637. 24. Misra, H.S., Khairnar, N.P., Barik, A., Indira Priyadarsini, K., Mohan, H. and Apte, S.K., 2004. Pyrroloquinoline quinone: a reactive oxygen species scavenger in bacteria. FEBS letters, 578(1-2), pp.26-30. 25. Paulino-Lima, I.G., Fujishima, K., Navarrete, J.U., Galante, D., Rodrigues, F., Azua- Bustos, A. and Rothschild, L.J., 2016. Extremely high UV-C radiation resistant microorganisms from desert environments with different manganese concentrations. Journal of Photochemistry and Photobiology B: Biology, 163, pp.327-336. 26. Peng, F., Zhang, L., Luo, X., Dai, J., An, H., Tang, Y. and Fang, C., 2009. Deinococcus xinjiangensis sp. nov., isolated from desert soil. International journal of systematic and evolutionary microbiology, 59(4), pp.709-713. 27. Prazdnova, E.V.E., Dem'yanenko, S.V., Chistyakov, V.A.E., Lysenko, V.S. and Batyushin, M.M., 2014. Carotenoids-antioxidants of D. radiodurans stimulate regeneration in mice. Biology and Medicine, 6(3), p.1. 28. Saito, T., Ohyama, Y., Ide, H., Ohta, S. and Yamamoto, O., 1998. A carotenoid pigment of the radioresistant bacterium Deinococcus radiodurans. Microbios, 95(381), pp.79-90. 29. Scarpa, E.S. and Ninfali, P., 2015. Phytochemicals as innovative therapeutic tools against cancer stem cells. International journal of molecular sciences, 16(7), pp.15727-15742. 30. Singh, O.V. and Gabani, P., 2011. Extremophiles: radiation resistance microbial reserves and therapeutic implications. Journal of applied microbiology, 110(4), pp.851-861. 31. Singh, S.P., Klisch, M., Sinha, R.P. and Häder, D.P., 2010. Genome mining of mycosporine-like amino acid (MAA) synthesizing and non-synthesizing cyanobacteria: a bioinformatics study. Genomics, 95(2), pp.120-128. 32. Singleton, V.L., Orthofer, R. and Lamuela-Raventós, R.M., 1999. Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. Methods in enzymology, 299, pp.152-178. 33. Stahl, W. and Sies, H., 2003. Antioxidant activity of carotenoids. Molecular aspects of medicine, 24(6), pp.345-351. 34. Sun, Z., Shen, S., Tian, B., Wang, H., Xu, Z., Wang, L. and Hua, Y., 2009.

Page 200

Functional analysis of γ-carotene ketolase involved in the carotenoid biosynthesis of Deinococcus radiodurans. FEMS microbiology letters, 301(1), pp.21-27.

35. Tamura, K. and Nei, M., 1993. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular biology and evolution, 10(3), pp.512-526. 36. Tamura, K., Stecher, G., Peterson, D., Filipski, A. and Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular biology and evolution, 30(12), pp.2725-2729. 37. Tatsuzawa, H., Maruyama, T., Misawa, N., Fujimori, K. and Nakano, M., 2000. Quenching of singlet oxygen by carotenoids produced in Escherichia coli–attenuation of singlet oxygen mediated bacterial killing by carotenoids. FEBS letters, 484(3), pp.280-284. 38. Terao, J., 1989. Antioxidant activity of β-carotene-related carotenoids in solution. Lipids, 24(7), pp.659-661. 39. Tian, B., Sun, Z., Shen, S., Wang, H., Jiao, J., Wang, L., Hu, Y. and Hua, Y., 2009. Effects of carotenoids from Deinococcus radiodurans on protein oxidation. Letters in applied microbiology, 49(6), pp.689-694. 40. Tian, B., Xu, Z., Sun, Z., Lin, J. and Hua, Y., 2007. Evaluation of the antioxidant effects of carotenoids from Deinococcus radiodurans through targeted mutagenesis, chemiluminescence, and DNA damage analyses. Biochimica et Biophysica Acta (BBA)- General Subjects, 1770(6), pp.902-911. 41. Wang, L., Min, M., Li, Y., Chen, P., Chen, Y., Liu, Y., Wang, Y. and Ruan, R., 2010. Cultivation of green algae Chlorella sp. in different wastewaters from municipal wastewater treatment plant. Applied biochemistry and biotechnology, 162(4), pp.1174-1186. 42. Wang, L., Min, M., Li, Y., Chen, P., Chen, Y., Liu, Y., Wang, Y. and Ruan, R., 2010. Cultivation of green algae Chlorella sp. in different wastewaters from municipal wastewater treatment plant. Applied biochemistry and biotechnology, 162(4), pp.1174-1186. 43. Xu, J., Chen, S. and Hu, Q., 2005. Antioxidant activity of brown pigment and extracts from black sesame seed (Sesamum indicum L.). Food chemistry, 91(1), pp.79-83. 44. Yu, L.Z.H., Luo, X.S., Liu, M. and Huang, Q., 2015. Diversity of ionizing radiation resistant bacteria obtained from the Taklimakan Desert. Journal of basic microbiology, 55(1), pp.135-140. 45. Zhang, P. and Omaye, S.T., 2000. β-Carotene and protein oxidation: effects of ascorbic acid and α-tocopherol. Toxicology, 146(1), pp.37-47.

Page 201

46. Zhang, Y.Q., Sun, C.H., Li, W.J., Yu, L.Y., Zhou, J.Q., Zhang, Y.Q., Xu, L.H. and Jiang, C.L., 2007. Deinococcus yunweiensis sp. nov., a gamma-and UV-radiation-resistant bacterium from China. International journal of systematic and evolutionary microbiology, 57(2), pp.370-375. 47. Zhishen, J., Mengcheng, T. and Jianming, W., 1999. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food chemistry, 64(4), pp.555-559.

48. i i , ., erpen, ., o u, G., G en, . an ančetovi , ., 2012. Phenolic compounds, carotenoids, anthocyanins, and antioxidant capacity of colored maize (Zea mays L.) kernels. Journal of Agricultural and food chemistry, 60(5), pp.1224-1231.

Page 202

Chapter 6: Ectoine a Compatible Solute in Radio-halophilic Bacteria

Paper 4

Title: Ectoine: a Compatible Solute in Radio-halophilic Stenotrophomonas sp. WMALM19 Strain to Prevent Ultraviolet-Induced Protein Damage

In review Journal of Applied Microbiology

Page 203

6.1. Abstract

Ectoines are amorphous water-binding molecules that form large hydrate envelopes to stabilize the natural structure of biopolymers and other vital biomolecules. The purpose of this study was to assess the antioxidant and radioprotective properties of a compatible solute, ectoine, extracted and purified from radio-halophilic bacterium. Nine different radio-resistant bacteria were isolated from desert soil, where strain WMA-LM19 was selected on the basis of its high tolerance for ultraviolet radiation among all these isolates. 16S rRNA gene sequencing indicated the bacterium was closely related to Stenotrophomonas sp. (KT008383). A bacterial milking strategy was applied for extraction of intracellular compatible solutes in 70% ethanol, purified by high performance liquid chromatography (HPLC) using a C18 analytical column. The antioxidant and radio-protective properties of the pure compound were evaluated by hydroxyl scavenging, DPPH reducing, lipid peroxidation inhibition assays and protein radio-protection assays with SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) analysis. The compound was characterized as ectoine by 1H and 13C NMR (Nuclear Magnetic Resonance), and mass spectrometry (MS). Ectoine exhibited strong Fe2+ chelation in comparison to EDTA (38.58± 0.846%). The OH- radical scavenging efficiency of ectoine (53.68 ± 0.48863%) was estimated in terms of % inhibition of deoxy D-ribose degradation in a non-site-specific assay using a concentration of 10.0 μg/mL. Maximum reduction in DPPH (~60.45 ± 1.185%) was observed at 10 μg/mL ectoine concentration. Ectoine effectively inhibited oxidative damage to proteins and lipids in comparison to the standard ascorbic acid. Furthermore, a high level of ectoine-mediated protection of bovine serum albumin against ionizing radiation (1500-2000 Jm-2) was observed, as indicated by SDS-PAGE analysis. The results indicated that ectoine can be used as a potential mitigator and radio- protective agent to overcome radiation- and salinitymediated oxidative damage in extreme environments.

Keywords: Extremophiles, Ectoine, Antioxidant, Sun screen, Nuclear Magnetic Resonance 6.2. Introduction

A capability of microorganisms to regulate osmotic pressure is a critical consideration when evaluating their ability to successfully compete and grow in a given habitat. This unique feature of extremophiles facilitates extensive applications in biotechnology, ranging from bioremediation of contaminated composites to the assembly of medicinal important drugs (Gabani et al., 2013). Most living cells have to adapt to fluctuations in the osmotic strength of their environment within certain limits. The proliferation of extremophiles in a given habitat, is strictly dependent upon their ability to maintain an internal osmotic pressure. In order to maintain an osmotic equilibrium, these microbes in extreme environment (bacteria, archea and eukarya) can equilibrate to saline environments by accumulating compatible solutes (Csonka et al., 1991). The diverse chemical structures of compatible solutes range from sugars, polyols to amino acid derivatives, and are not only considered as osmoregulatory solutes, but also valuable to bacterial cells in protecting proteins from the damaging effects of drying, freezing, high temperatures and ultraviolet radiations (Kunte et al., 2014; Ventosa and Nieto, 1995).

Page 204

a) Structure of Betaine b) Glycine zwitterion c) Structure of glycine

Figure 6.1: Structure of a) betaine b) glycine zwitterion c) glycine, compatible solutes from extremophiles.

The compatible solute ectoine (1,4,5,6-tetrahydro-2-methyl-4- pyrimidinecarboxylic acid) is a cyclic amino acid derivative of aspartate, which has achieved extensive interest as a protective agent, and appears to be a universal organic solute in bacteria. It was originally discovered in the phototrophic, halophilic bacterium Ectothiorhodospira halochloris (Galinski et al., 1985). Ectoine is considered one of the most effective protectants of nucleic acid, proteins, cell membrane and even whole cells against freezing, heating, drying or other chemical agents (Louis et al., 1997; Severin et al., 1992). Action biosynthesis has been documented in a great variety of halotolerant and halophilic species, particularly in those with simple growth demands. Ectoine biosynthesis is far more widespread in the bacterial domain than betaine and glycine (Oren, 2008). Ectoine is currently produced and has extensive applications in cosmetic additives, biological processes and enzyme preparations, pharmaceutical industries and other fields for commercial usage (Kanapathipillai et al., 2008; Lentzen and Schwarz, 2006; Zhang et al., 2006). There is hence great interest in the technology that leads to the efficient production of ectoine. Abundantly halophilic and halotolerant microorganism were investigated that could produce ectoine in salt stress environments (Pastor et al., 2010). The bacterial process for production of ectoine has been modified in the past two decades to meet increasing market demands. The yield of ectoine can be maximized by a technical bioprocess called “bacterial milking” using the halophilic eubacterium H. elongata (Sauer et al., 1998), which involves a cyclic upturn and diminution of the salt concentration for ectoine synthesis and secretion, correspondingly. Ectoines and their derivatives, like hydroxy ectoine, are now being mainly produced biologically widely. These compatible solutes are used in cosmetics as anti-aging agents, for oral care and as adjuvants for vaccines (http://www.bitop.de; http://www.merck.de). In addition, these solutes are not only limited to use as stress protectants, but can serve as an important source of carbon and nitrogen for energy production (Salar-García et al., 2017) intracellularly, and upon release to the surrounding medium, can induce cell death or hypo- osmotic shock in other micro-organisms.

Ectoine’s protective properties of proteins can be explained by its strong osmotropic interaction with water and subsequent exclusion from protein surfaces, the strengthening of intra-molecular hydrogen bonding (secondary structures) and the decrease of solubility of the peptide backbone (Kunte et al., 2014). Ectoine as a novel active component in health care products and cosmetics has attracted industry because of its stabilizing and UV protective properties. The halophilic bacterium Halomonaselongata has been used as a producer strain in joint efforts of industry and research to develop large-scale fermentation procedures (Hahn et al., 2015). Bacterial milking procedure and the development and application of ectoine excreting mutants

Page 205

(“leaky mutants”) are the two key technologies that allow the annual production of ectoine on a scale of tons. Despite of being more successful method bacterial milking also has some drawbacks. This method is inexorable with the decline in cell growth rate, corrosion of equipment, and the difficulty of downstream handling due to the discontinuous production pattern and high salt mediation (Schubert et al., 2007).

To overcome these shortages, Stenotrophomonas sp. strain WMA-LM19, a UV resistant strain isolated from desert soil, a halophilic bacterium that can nurture and excrete ectoine in high manganese and salt concentration, was chosen for ectoine production using monosodium glutamate as a carbon and nitrogen source in 0.5M NaCl. Ectoine was purified and their possible protective role as an antioxidant to protect the cellular protein in stress was also evaluated. Protective effect offered by ectoine to BSA and red bold cells membrane was also evaluated using SDS-PAGE and membrane damage assay. 6.3. Materials and Methods

6.3.1. Bacterial Strain and Growth Conditions Soil samples were collected aseptically in sterile zipper plastic bags from the dessert of District Lakki Marwat, Khyber Pukhtoonkhwa, Pakistan, and kept at 4°C until further investigation for radio-resistant microbial community. Serially diluted soil sample were inoculated by spread plate method on Spizizen's minimal medium (SMM) medium containing: (NH4)2SO4 (2 g/L); Na- citrate.2H2O (1 g/L); K2HPO4 (14 g/L); KH2PO4 (6 g/L), MgSO4.7H2O (0.2 g/L), MnCl2.4H2O (0.01g/L), and NaCl (30 g/L), with 0.5% glucose as the carbon source supplemented with phenylalanine (18 mg/L), and tryptophan (20 mg/L). SMM plates were exposed to UV radiation for 5 min in UV chamber (119x69×52 cm) supplied with a 20W, 280 nm UV light, prior to incubation. The UV fluency rate was calculated as He = Ee×t in units of J/m2. The isolates were subcultured from irradiated plates after incubation at 37°C for 5-7 days.

6.3.2. Bacterial Survival Curves at UVB and Oxidative Stress Strain WMA-LM19 was tested for radiation resistance and survival curve was plotted (Mattimore and Battista, 1996). Cells were serially diluted with phosphate buffer saline and spread on SMM agar plates, then exposed to different doses of UV radiation at 280 nm. The survival rate was determined by dividing the number of colonies appeared on UV-irradiated plates with colonies from un-irradiated culture. 6.3.3. Identification of Radioresistant Bacterium Strain WMA-LM19 was identified morphologically as well as biochemically by the method as previously described (Murray et al., 1981). 16S rRNA gene was sequenced for molecular identification of strain WMA-LM19. DNA was extracted by DNA extraction kit (QIAGEN) and 16S rRNA gene sequence was amplified using 27F’ (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R’ (5’- CTACGGCTACCTTGTTACGA-3’) bacterial primers. The amplified PCR product was sequenced by Macrogen Service Center (Geunchun-gu, Seoul, South Korea). The sequence obtained was computed for nearest relatives in the NCBI database using BLAST tool and homologues were analyzed for phylogeny using Molecular Evolutionary Genetic Analysis (MEGA) version 6. The neighbour-joining phylogenetic tree was constructed for the

Page 206

identification of isolated bacterium and diversity among UV resistant extremophiles was studied. The sequence was submitted to NCBI GeneBank in order to assign an accession number. 6.3.4. Extraction and Determination of Ectoine by NMR Spectroscopy Extraction of intracellular compatible solutes was carried out by the method as previously described with slight modification (Wang et al., 2006). Strain WMALM19 was grown in 200 ml SMM at a high salt concentration [8 % (w/v) NaCl] to induce the accumulation of ectoine. When the optical density (OD600) was reached up to 1 (1 OD/mL = 0.31 g DCW/L), cells were collected by centrifugation at 1000 x g for 10 min at 4°C and washed with 50mM potassium phosphate buffer (pH 7.2) containing isotonic NaCl concertation. The cells were transferred to a deionized water of low salinity [hypo-osmotic shock from 8% to 3% (w/v) NaCl], the accumulated solutes within the cells were rapidly released to achieve the osmotic equilibrium. These solutes were extracted by re-suspending the pellets in 80% ethanol for 10 hr and then centrifuged. After the centrifugation 1 supernatant was collected, dried and dissolved in 0.8 ml methanol-d4 (CD3OD) for proton ( H) and carbon (13C) NMR. NMR spectroscopic data were recorded at ambient temperature on Bruker

AMX-400 MHz in CDCl3 with tetramethylsilane (TMS) as an internal standard (temperature of 20- 22°C).

For HPLC the extract was further purified by using whatman filter paper, applying a second crossflow filtration in a mixture of water and ethanol. 6.3.5. High Performance Liquid Chromatography (HPLC) Analysis of Ectoine The cellular extract was filtered using a small pore size membrane filter (0.22 m) and subjected to isocratic HPLC (Agilent 1260 series, Hewlett-Packard) C18 (2.6 × 250 mm, 5 um). The extract was eluted with a mixture of methanol/water (70:30) as mobile phase for 10 minutes. The flow rate was adjusted to 0.8 ml/min using UV detection at 210 nm to measure ectoine and confirmed by LC-MS/MS. 6.3.6. Liquid Chromatography–Mass Spectrometry LC-MS Analysis of Ectoine The m/z (mass to charge ratio) was determined by using triple quardrupole mass spectrometer with Electro-Spray Ionization (ESI) source in positive mode ionization (Agilent, USA). The compound was eluted with a mixture of water and methanol (5/95%) containing formic acid (0.1%) with an isocratic conditions at a flow rate of 0.8 mL/min and mass scan ranges from m/z 80-1500. The capillary voltage and atomizing gas pressure were 4.0 kV and 35 psi, respectively. The flow rate of drying gas (nitrogen as collision gas) was 12 mL/min and the temperature of solvent removal was 350°C.

6.3.7. Evaluation of Antioxidants and Radio-Protective Properties of Ectoine 6.3.7.1.

Hydroxyl radical scavenging activity estimation

The hydroxyl scavenging activity of ectoine was carried out by fenton method described by Halliwell and Gutteridge (1987). 0.1mM of ferric chloride solution was mixed with different concentrations of ectoine (2-10 g) previously reacted with 3.6 mM 2-deoxy-D-ribose, 0.1 mM L- ascorbic acid along with 1 mM H2O2 and 0.104 mM EDTA-Na in potassium phosphate buffer (pH 7.4) in a total assay volume of 1 ml followed by incubation at room temperature for 1 hr. The

Page 207

reaction mixture was then treated with 10% TCA and 0.5% thiobarbituric acid (in 0.025M NaOH), incubated for 15 min at 55ºC. The resulted pink chromogen generated from degradation of deoxyribose by OH was measured at 532 nm and scavenging potential of ectoine was calculated by following formula.

% Inhibition=[(Abscontrol – Abstest)/Abscontrol]x100

6.3.7.2. Radical Neutralizing Activity by DPPH Assay The free radical scavenging ability of ectoine was based on decay of DPPH (2,2-Diphenyl-1- Picrylhydrazyl) (Xu et al., 2005). Different concentration of ectoine ranges from 2-10 g was added to 1.0 ml of DPPH solution (100 μM). The mixture was incubated at room temperature for 30 min. using ascorbic acid as positive control (Rao et al., 2006). The absorbance was taken at 517 nm and percent scavenging activity of ectoine was calculated according to the formula

%Inhibition = (Abscontrol – Abstest)/Abscontrol x 100

6.3.7.3. Lipid peroxidation inhibition assay Thiobarbituric acid reactive substances assay was carried out for lipid peroxidation by using lipid rich medium (mice liver homogenate) (Ohkawa et al., 1979). 1 gm of liver tissue was homogenized in tris HCL buffer of pH 7.4 and centrifuged at 3000 ×g for 5 minutes. 1 mL of the homogenized mixture was mixed with different concentration ranges from 2-10 g of ectoine. 10 M FeSO4 was added and the volume was made up to 0.3 ml. 0.2 ml of SDS (8.1%), 0.5ml acetic acid (pH 3.4) and an equal volume of thiobarbituric acid (0.6% v/v) were added and incubated for 1 hr at 100oC. The absorbance of colored mixture was measured at 532 nm and the absorbance was compared with that of a standard curve using malondialdehyde (MDA) using the blank without adding FeSO4. 6.3.7.4. Protein Oxidation Inhibition Assay The inhibitory effect of ectoine on protein oxidation in-vitro was quantified using bovine serum albumin as standard proteins (Tian et al., 2009). Different concentration of ectoine (2-10 g) was incubated with 200 l of BSA (target protein).

The mixture was then treated with 100 l of oxidant FeSO4 (1 mm), 100 l H2O2 (80mm) and incubated for 1hr at 37oC. The reaction was stopped by adding 15 U of catalase. 10mmol/l of DNPH was added to the mixture and incubated for 1hr, the unbound proteins to DNPH were precipitated by adding 10% TCA. The supernatant was dissolved in guanidine hydrochloride (6M) and protein-oxidation was quantified spectrometrically at 370 nm using H2O to replace FeSO4 and

H2O2 as blank. Percent inhibition offered by ectoine was calculated using following formula.

(%) Inhibition of protein oxidation= (Control-sample/control) x 100

6.3.7.5. Assay of Reducing Power The reducing potential of ectoine was determined by the method described by Oyaizu (1986). Ectoine in different concentrations ranges from (0-10.0μg/mL) was mixed with 1 ml of o ferricyanide [1% K3Fe(CN)6] and 0.1mM phosphate buffer (pH 7.0), incubated at 50 C for 20min. 10% TCA was added to mixture to stop the reaction. The supernatant was removed and mixed with FeCl3 (0.5 mL, 0.1% w/v). % reducing potential was calculated my measuring the absorbance at 700 nm using the formula (A0-A1)/A0 × 100.

Page 208

6.3.7.6. Iron (Fe+2) Chelating Activity The chelation potential of ectoine ranges in different concentration (010.0μg/mL) was determined by the method as described previously (Dinis et al., 1994). 50 μl of the sample was mixed with a solution of FeCl2 (2mM). The rection was initiated by adding 50μl of ferrozine (5mM). The mixture was incubated at room temperature for 10 minutes. The optical density was measured at 562 nm once the reaction had reached equilibrium. %chelating activity of ectoine was calculated using the formula.

Chelating activity (%) = (A0- A1)/A0 × 100. EDTA was used as positive control. 6.3.7.7. Membrane Protection Assay Human red blood cells (erythrocytes) were isolated by centrifugation of citrated blood at 2000xg for 15 minutes. Cells were washed with phosphate buffer saline and then re-suspended in the same buffer to the desired hematocrit level (Yang et al., 2006). Cells were pre-treated with Ectoine (1%) and lecithin (1%), in separate, and then kept under stress for 1 hr at 37oC in the presence of surface damaging substances like sodium dodecyl sulfate, alkylpoly glucoside, Na- Laurylethersulfate, benzalkoniumchloride. 200 μl of the reaction mixture was taken and centrifuged at 3000×g for 5minute. The supernatant was collected and absorbance was determined at 540 nm. The percentage haemolysis was calculated by following formula:

% haemolysis = (control-test/control) x100

6.3.7.8. Analysis of Radio-Protection to BSA by Ectoine The structural damage to BSA in response to different doses of irradiation (1500-2000 Jm-2) and protection in presence of ectoine was confirmed by SDS-PAGE analysis. The analysis was performed by preparing 0.75 mm thick polyacrylamide gel (10%). Protein was irradiated with different UV doses in the presence and absence of ectoine, then added to PAGE loading buffer [0.0625M Tris/HCl, pH 6.8, 5% (v/v) glycerol, 2% (v/v) 3-mercaptoethanol and 0.01% (w/v) Bromophenol Blue]. Sample was loaded on 10% polyacrylamide gel (0.75mm) and electrophoresis was carried out at constant voltage (90v). The gel was stained in 0.1% Coomassie Brilliant Blue R250 by gentle shaking at room temperature for 1 hr and detained in methanol/acetic acid/water mixture (4:2:4), bands were observed over a clear background. 6.4. Results:

A total of 09 different radio-resistant bacterial strains were isolated, strain WMA-LM19 was selected on the basis of high resistance to UV radiation among all these isolates, i.e., 50% survival rate to UV dose (280 nm) was 5 ×103 Jm-2.

6.4.1. Resistance to UV radiation, oxidative stress and Mitomycin C Strain WMA-LM19 was exposed to different energy doses of UV radiation in order to determine its radiation resistant potential. Strain WMA-LM19 maintained nearly 50% viability at 1300 Jm-2 energy dose of UV radiation, whereas the E. coli (10536) couldn't survive at such high energy UV radiation (Fig. 6.2A). A gradual decrease in survival of strain WMA-LM19 was observed to increase in concentration of H2O2 and maintained up to 50% viability at 6mM H2O2 for 60min (Fig. 6.2B). Results are expressed as means ± SD and are compared using the Student’s unpaired t-test. Moreover, the percentage values had an exponential distribution. Error bars represent standard deviation for triplicate experiments. P value < 0.05 is considered significant.

Page 209

6.4.2. Identification of Strain WMA-LM19 Strain WMA-LM19 was aerobic, gram-negative rod shaped and grown white to light pink, raised, mucoid colonies. 16S rRNA sequence of strain WMA-LM19 was assembled by DNA baser software and subjected to blast search in National Centre for Biotechnology Information (NCBI). The results indicated that strain belongs to genus Stenotrophomonas with 93% similarity. The phylogenetic tree was constructed and WMA-LM19 was clustered into Proeteobacteria group among the sequences obtained from NCBI. The nucleotide sequence reported here can be obtained from NCBI nucleotide sequence database under accession number KT008383.

Page 210

Figure 6.2: Survivability of strain WMA-LM19 from desert soil at varying UV-B exposure. (A) UV radiation resistant potential of WMA-LM19 (B) Resistance to different concentrations of hydrogen peroxide (mM). % viability value is calculated as N1/N0x100 where Ni is the value after exposure to irradiation and H2O2 while N0 is the value at time 0, for each condition tested. Results are highly significant different in among group as p value is less than significant level 0.05. Values are mean ± SD.

Figure 6.3: Neighbor joining phylogenetic tree based on 16S rRNA gene sequence analysis, showing the position of isolate WMA-LM19 to other strains of Stenotrophomonas obtained from NCBI. Accession numbers of the sequences used in this study are shown in parentheses after the strain designation. Numbers at nodes are percentage bootstrap values based on 1,000 replications.

Page 211

Figure 6.4: HPLC chromatogram/positive of the polar extract for WMA LM19, that exhibit signal at 210 nm with a retention time of 3.0 minutes. 6.4.3. Purification and Identification of Compatible solute from strain WMA- LM19

6.4.3.1. LCMS analysis of the extract

The ethanol extract of compatible solute from Stenotrophomonas sp. strain WMA-LM19 exhibited a single peak on HPLC at 3.0 minutes retention time (Fig. 6. 4). For further confirmation the extract was subjected to LCMS on an ABX3200 QTRAP mass spectrometer equipped with a TurbolonSpray ESI source, and connected to a Shimadzu HPLC system with dual LC-20 pumps, a SPD-M20A UV/Vis photodiode array (PDA) detector and autosampler. Mass data were collected in positive ionization mode and the mass scan width was set to m/z 100-1700. This peak showed a mass of 143.68 by ESI/MS, and matched with the formula

C6H10N2O2 (Fig. 6.5) and was identified as ectoine by 1H and 13C NMR.

6.4.3.2. 1H and 13C NMR

NMR data were acquired on a Bruker Avance 500 NMR spectrometer in XXX solvent, referenced 1 to residual protonated solvent ( H 3.35 ppm, C 49.3 ppm) Representative H NMR spectra for the extract from the bacterial cells (Fig 6.6) showed the presence of diastereotopic protons in the compound, which together with the N-H singlet at H 2.2 ppm, was consistent with the structure of ectoine. The 1H NMR data were found to be identical to those reported in the literature for 13 ectoine, as were the C NMR data (Fig 6.7). The presence of signals at C 161.671 (C-CH3) and 175.993 (C=O) in the 13C spectrum confirmed the presence of a carboxyl group, while signals at 18.7, 23.1, 38.7, and 55.0 ppm showed the 2-CH and 3-CH aliphatic carbons in ectoine. Thus, on the basis of the NMR and LC-MS data, ectoines were accumulated in the cells.

Page 212

Figure 6.5: ESI-MS spectrum of purified ectoine extract exhibits signals at m/z 143.2036 [M+H]+.

Figure 6.6: 1H NMR spectra of purified compound LM19F2.

Page 213

Figure 6.7: 13C NMR spectra of purified compound LM19F2. The purified compound was identified as “Ectoine”

Figure 6.8: Chemical structure of ectoine from NMR spectroscopy.

Page 214

6.4.4. Antioxidants and Radio-Protective Properties of Ectoine 6.4.4.1. DPPH and Hydroxyl Radical Scavenging Potential DPPH is a stable radical and appears as violet colour solution. Any compound with good antioxidant activity can reduce DPPH and resulted disappearance of the characteristic violet colour. Concentration dependent decrease in absorbance of DPPH solution by ectoine was noticed. Ectoine mediated DPPH reduction up to 60.45±1.1876% at 10 μg/ml concentration. This concentration was found highly comparable to the positive control ascorbic acid in this study (10 μg/ml) as shown in figure 6.9. The scavenging potential of hydroxyl radical with ectoine was evaluated by % deoxyribose degradation inhibition. Maximum % inhibition in deoxyribose degradation was observed as 53.6879±1.1856% at 10 μg/mL concentration of ectoine (Fig. 6.9).

6.4.4.2. Reducing capacity and Fe2+ chelating activity The reducing and Fe chelating potential of ectoine was increased with increase in its concentration. Ectoine demonstrated a significant reducing and chelation activity than those of ascorbic acid and EDTA, both of them were considered as positive control (Fig. 6.10). The reducing capacity and Fe2+ chelating activities were 53.81% ± 0.531 and 49.49% ± 0.3068 at concentrations of 10.0 μg/mL, respectively in comparison to ascorbic acid (30.78%) and EDTA (38.59%) (Fig. 6.10). 6.4.4.3. Lipid peroxidation and protein carbonylation inhibition Ectoine showed a concentration dependent inhibitory effect on lipid peroxidation and protein carbonylation/oxidation. The oxidative damage to lipid (mice liver homogenate) and proteins (BSA) was inhibited to 66.01% and 72.09%, respectively at 10 μg/mL of ectoine that was comparatively higher than ascorbic acid (55.45%) (P > 0.05). The study demonstrated a significant effect on protein and lipid oxidative damages in the presence and absence of ectoine (Fig. 6.11).

6.4.4.4. Membrane protection A marked protection was provided by ectoine against all kind of surface membrane-damaging active substances used in the membrane protection assay. Ectoine demonstrated more efficient preventive activity (54.80%) to erythrocytes membrane in the presence of Benzalkoniumchloride in comparison to 1% lecithin (28.915%) that serves as a positive control (Fig. 6.12). 6.4.4.5. Protective efficacy of ectoine against radiation induced damage An aqueous solution of bovine serum albumin (BSA) was irradiated with different doses of UV in the presence of ectoine and protein was analyzed by SDSPAGE, its breakdown was observed in terms of intense smeared band. The radiation dose dependent (1500-2000 Jm-2) degradation of BSA was observed in the form of intense smeared bands appeared on SDS-PAGE as compared to untreated control. However, no smearing of BSA bands was found upon irradiation (1500-2000 Jm-2) in the presence of ectoine. The results clearly demonstrate significantly high radioprotective efficacy of ectoine towards BSA against supra lethal ionizing radiation induced damage (Fig. 6.13).

Page 215

Figure 6.9: DPPH and OH Free radical scavenging assay using ascorbic acid as positive control. The reduction of the DPPH radicals was estimated as the function of decrease absorbance as monitored at 517 nm.

Figure 6.10: Assay of reducing power and Fe chelation of ectoine using ascorbic acid and EDTA as positive control. Ectoine exhibited significant % inhibition.

Page 216

Figure 6.11: Protein oxidation and lipid peroxidation inhibition activity offered by ectoine using bovine serum albumin (standard protein) and mice lipids as test samples.

Page 217

Figure 6.12: Membrane damage preventing assay by ectoine using human RBCs and Lecithin as positive control to different membrane damaging substances.

Page 218

Figure 6.13: Analysis of protein protection offered by ectoine on SDS-PAGE. Observance of protein protection (BSA) offered by ectoine (25 μg/ml) at radiation doses 1500- 2000 Jm-2.

6.5. Discussion

Strain WMA-LM19 (KT008383) was found to produce ectoine intracellularly under UV radiation and salt stress via bacterial milking strategy. This result was confirmed by the HPLC, LC–MS analyses as well as NMR data. Furthermore the inhibitory role of ectoine against protein and lipid

Page 219

oxidative damage was also determined. To the best of our knowledge, there are limited reports on ectoine production from wild-type strain of radio-resistant halophilic bacteria.

Among total 09 bacterial strains isolated from desert soil, strain WMA-LM19 was found to grow under high UV dosage and salt concentration (15%). The results of 16S rRNA gene sequencing indicated that strain WMA-LM19 showed 93% sequence similarity to Stenotrophomonas genus. Strain WMA-LM19 accumulates high concentration of ectoine intracellularly in the presence of manganese chloride, high salinity and UV radiation. The slight change in morphology such as an increase in colony size might be due to high concentration of Mn. Accumulation of Mn and high ectoine might be two interesting mechanisms of survival under high UV radiation and salinity, as Mn+2 block the Fenton reactions and quench superoxides, thus preventing the cells from oxidative damage. On the other hand, ectoine act as an anti-oxidant and preventing cells from desiccation and water loss. In this report, the productivity of ectoine from ectoine-non excreting type strain Stenotrophomonas strain WMA-LM19 was 2.9 gl-1d-1, which is the highest level of ectoine productivity. Sauer and Galinski (1998) investigated the production of ectoine 3.3 g l-1d-1 from Halomonas elongata (an ectoine excreting strain) DSM 142 using bacterial milking strategy. While Brevibacterium epidermis DSM 20659 an anaerobic denitrifying halophilic bacterium capable of producing ectoine up to 2 g l-1d-1 (Onraedt et al., 2005).

HPLC analysis of the medium reveals no peak after centrifugation and filtration of the cell pellets, demonstrating that ectoine is produced intracellularly preventing the excessive loss of water from the cells in extreme environment of high salinity and UV radiations. Ectoine from strain Stenotrophomonas sp. WMA-LM19 was investigated for its role in cellular protection against radiation mediated oxidative damage. Hydroxyl (OH-) radical is the most reactive oxygen formed during the reaction of transition metals with various peroxides. These OH-radicals ultimately attack on cellular macromolecules such as DNA, poly unsaturated fatty-acids and proteins, thus damaging the cells (Aruoma et al., 1999). In our findings ectoine demonstrated a significant scavenging potential of non-site specific hydroxyl ion. The presence of ectoine makes the Fe un- available for attacking deoxyribose (Kitts et al., 2000). Our results demonstrated that ectoine is a strong iron chelator. Chelating agents may protect DNA from hydroxyl radicals by protecting enzymes responsible for DNA repair. Tissues enriched with iron containing proteins readily releases iron and copper ions in the form that are capable of catalysing such free radical formation, lipid peroxidation and autoxidation of neurotransmitters (Spencer et al., 1994). Iron chelation therapy leads to low cytosolic iron concentrations that facilitate resistance by protecting proteins, more than DNA, from IR-induced oxidative damage. By three folds decrease in iron content in some bacterial species may lead to increase in radioresistance up to 2000 times than human lethal dose (Daly, 2009; Flora et al., 2010).

There seems to be a direct relationship between quenching ability of ectoine for hydroxyl radicals and prevention of lipid peroxidation process. DPPH assay is the most efficient tool to evaluate the antioxidant properties of any compound. The antioxidants react with free radicals and extract electrons from it to get reduced. We investigated that ectoine extracted and purified from Stenotrophomonas sp. WMALM19 offered an efficient scavenging activity of DPPH radical in a concentration dependent manner. The two fold stronger quenching abilities of ectoine may be the results of keto group that can quench the super-oxides and thus preventing the cell from photo-oxidation and excessive damages in extreme environments (Buenger et al., 2004; Ventosa et al., 1998).

Page 220

As antioxidant potential has been recognized as the contributory factor to radio-protection offered by any compound, ectoine-mediated radio-protection to BSA was evaluated by performing native PAGE. A clear, intense smearing of BSA was observed as a result of oxidative damage to its secondary structure due to increase in radiation dosage in the absence of ectoine. However, no smearing was observed in the presence of ectoine that provides enough evidence of ectoine-mediated UV protection. Ectoine treatment to BSA results into formation of relatively more stable product, which in turns contributes to inhibition of oxidative damages in proteins and lipids in stress condition (Cheng et al., 2014). The kosmotropic nature of ectoine decreasing the solubility of peptide back bone and strengthens the intramolecular hydrogen bonding or secondary structures thereby offering a significant protection to proteins. Furthermore, ectoine and other compatible solutes effect on preventing aggregation of amyloidogenic proteins by stabilizing the native state (Arora et al., 2004; Botta et al., 2008; Furusho et al., 2005). Ectoines have considerable industrial and pharmacological interest due to function preservation and stabilizing effects (Pastor et al., 2010). Ectoine and their derivatives have gained significant commercial market value. Recent biotechnological techniques via “bacterial milking” enhanced the annual ectoine production and reached the scale of tons (Sauer et al., 1998; Schwibbert et al., 2011).

Concerning the interaction with other biomolecules, as for example, lipids of the cytoplasmic membrane, research on native fluid systems (as apposed to dry stabilization of liposomes) are still at the beginning. Ectoine also stabilizes the membranes of pre-treated cells (erythrocytes from human RBCs) against the damaging effect of stress factors and surfactants as well that lead to dehydration (hemoglobin release). In the same way skin cells also have a double lipid layer configuration within targeted and linked proteins. Cell survival largely depends upon function of this membrane that control permeability, structure of molecules and ions in the cell by special ducts and pumps. A number of external parameters like temperature, pH, radicals and other chemical factors can disturb this equilibrium and damage the membranes. However, it has been shown that the cell membrane of pretreated cells are protected and stabilized by ectoine against damaging effect of surfactants and suggesting its kosmotropic nature. Thus, ectoine features long- term moisturizing efficacy as it is more potent moisturizer than glycerol and other membrane protective agent like lecithin (Graf et al., 2008). Ectoine attracted industry due to its UV protective and stabilizing properties with potential market value as active component in cosmetics and health care products (Bünger et al., 2001).

6.6. Conclusion

Based on the current study, it can be concluded that ectoine carried strong antioxidant properties, and thus can neutralize in vitro radiation-induced free radicals efficiently. Ectoine can be used as potential mitigator and radio-protective agent to overcome the radiation and salinity mediated oxidative damages in extreme environment. Further studies are required to evaluate the radio- protective efficacy of ectoine in vivo. It will certainly explore the future applications in sunscreens and other UV absorbing compounds to treat different skin infections due to radiation exposure.

Page 221

Acknowledgment

This research was supported by grants from Higher Education Commission of Pakistan under international research support initiative program (IRSIP). We also highly acknowledge Oregon State University for providing the opportunity to work in collaboration. Conflict of interest

No conflict of interest is associated with this work.

References: 1. Arora, A., Ha, C. and Park, C.B., 2004. Inhibition of insulin amyloid formation by small stress molecules. FEBS letters, 564(1-2), pp.121-125. 2. Aruoma, O.I., 1999. Free radicals, antioxidants and international nutrition. Asia Pacific journal of clinical nutrition, 8, pp.53-63. 3. Botta, C., Di Giorgio, C., Sabatier, A.S. and De Méo, M., 2008. Genotoxicity of visible light (400–800nm) and photoprotection assessment of ectoin, l-ergothioneine and mannitol and four sunscreens. Journal of Photochemistry and Photobiology B: Biology, 91(1), pp.24-34. 4. Buenger, J. and Driller, H., 2004. Ectoin: an effective natural substance to prevent UVA- induced premature photoaging. Skin pharmacology and physiology, 17(5), pp.232-237.

Page 222

5. Bünger, J., Degwert, J. and Driller, H., 2001. The protective function of compatible solute ectoin on the skin, skin cells and its biomolecules with respect to UV radiation, immunosuppression and membrane damage. IFSCC Mag, 4, pp.127-131. 6. Cheng, J., Zhang, Z., Zheng, Z., Lv, G., Wang, L., Tian, B. and Hua, Y., 2014. Antioxidative and Hepatoprotective Activities of Deinoxanthin-Rich Extract from Deinococcus radiodurans R1 against Carbon TetrachlorideInduced Liver Injury in Mice. Tropical Journal of Pharmaceutical Research, 13(4), pp.581-586. 7. Csonka, L.N. and Hanson, A.D., 1991. Prokaryotic osmoregulation: genetics and physiology. Annual Reviews in Microbiology, 45(1), pp.569-606. 8. Daly, M.J., 2009. A new perspective on radiation resistance based on Deinococcus radiodurans. Nature Reviews Microbiology, 7(3), pp.237-245. 9. Dinis, T.C., Madeira, V.M. and Almeida, L.M., 1994. Action of phenolic derivatives (acetaminophen, salicylate, and 5-aminosalicylate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers. Archives of biochemistry and biophysics, 315(1), pp.161-169. 10. Flora, S.J. and Pachauri, V., 2010. Chelation in metal intoxication. International journal of environmental research and public health, 7(7), pp.2745-2788. 11. Furusho, K., Yoshizawa, T. and Shoji, S., 2005. Ectoine alters subcellular localization of inclusions and reduces apoptotic cell death induced by the truncated Machado–Joseph disease gene product with an expanded polyglutamine stretch. Neurobiology of disease, 20(1), pp.170-178. 12. Gabani, P. and Singh, O.V., 2013. Radiation-resistant extremophiles and their potential in biotechnology and therapeutics. Applied microbiology and biotechnology, 97(3), pp.993- 1004. 13. Galinski, E.A., PFEIFFER, H.P. and Trueper, H.G., 1985. 1, 4, 5, 6 Tetrahydro 2 methyl 4 pyrimidinecarboxylic acid. European Journal of Biochemistry, 149(1), pp.135- 139. 14. Graf, R., Anzali, S., Buenger, J., Pfluecker, F. and Driller, H., 2008. The multifunctional role of ectoine as a natural cell protectant. Clinics in dermatology, 26(4), pp.326-333. 15. Hahn, M.B., Solomun, T., Wellhausen, R., Hermann, S., Seitz, H., Meyer, S., Kunte, H.J., Zeman, J., Uhlig, F., Smiatek, J. and Sturm, H., 2015. Influence of the compatible solute ectoine on the local water structure: implications for the binding of the protein G5P to DNA. The Journal of Physical Chemistry B, 119(49), pp.15212-15220.

Page 223

16. Halliwell, B. and Gutteridge, J.M., 1992. Biologically relevant metal iondependent hydroxyl radical generation An update. FEBS letters, 307(1), pp.108-112. 17. Kanapathipillai, M., Lentzen, G., Sierks, M. and Park, C.B., 2005. Ectoine and hydroxyectoine inhibit aggregation and neurotoxicity of Alzheimer's β amyloid. FEBS letters, 579(21), pp.4775-4780. 18. Kitts, D.D., Wijewickreme, A.N. and Hu, C., 2000. Antioxidant properties of a North American ginseng extract. Molecular and cellular biochemistry, 203(1), pp.1-10. 19. Kunte, H.J., Lentzen, G. and Galinski, E.A., 2014. Industrial production of the cell protectant ectoine: protection mechanisms, processes, and products. Curr Biotechnol, 3, pp.10-25. 20. Lentzen, G. and Schwarz, T., 2006. Extremolytes: natural compounds from extremophiles for versatile applications. Applied microbiology and biotechnology, 72(4), pp.623-634. 21. Louis, P. and Galinski, E.A., 1997. Characterization of genes for the biosynthesis of the compatible solute ectoine from Marinococcus halophilus and osmoregulated expression in Escherichia coli. Microbiology, 143(4), pp.1141-1149. 22. Mattimore, V. and Battista, J.R., 1996. Radioresistance of Deinococcus radiodurans: functions necessary to survive ionizing radiation are also necessary to survive prolonged desiccation. Journal of bacteriology, 178(3), pp.633-637. 23. Gerhardt, P., Murray, R.G.E., Costilow, R.N., Nester, E.W., Wood, W.A., Krieg, N.R. and Phillips, G.B., 1981. Manual of methods for general bacteriology. 24. Ohkawa, H., Ohishi, N. and Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical biochemistry, 95(2), pp.351-358. 25. Onraedt, A.E., Walcarius, B.A., Soetaert, W.K. and Vandamme, E.J., 2005. Optimization of ectoine synthesis through fed batch fermentation of Brevibacterium epidermis. Biotechnology progress, 21(4), pp.1206-1212. 26. Oren, A., 2008. Microbial life at high salt concentrations: phylogenetic and metabolic diversity. Saline systems, 4(1), p.2. 27. Qyaizu, M., 1986. Studies on products of browning reactions: Antioxidative activities of product of browning reaction prepared from glucosamine. Japan J Nutri, 44, pp.307-315. 28. Pastor, J.M., Salvador, M., Argandoña, M., Bernal, V., Reina-Bueno, M., Csonka, L.N., Iborra, J.L., Vargas, C., Nieto, J.J. and Cánovas, M., 2010. Ectoines in cell stress protection: uses and biotechnological production. Biotechnology advances, 28(6), pp.782- 801.

Page 224

29. Sal Salar-García, M.J., Bernal, V., Pastor, J.M., Salvador, M., Argandoña, M., Nieto, J.J., Vargas, C. and Cánovas, M., 2017. Understanding the interplay of carbon and nitrogen supply for ectoines production and metabolic overflow in high density cultures of

Chromohalobacter salexigens. Microbial cell factories, 16(1), p.23. 30. Sauer, T. and Galinski, E.A., 1998. Bacterial milking: a novel bioprocess for production of compatible solutes. Biotechnology and bioengineering, 57(3), pp.306-313. 31. Schubert, T., Maskow, T., Benndorf, D., Harms, H. and Breuer, U., 2007. Continuous synthesis and excretion of the compatible solute ectoine by a transgenic, nonhalophilic bacterium. Applied and environmental microbiology, 73(10), pp.3343-3347. 32. Schwibbert, K., Marin Sanguino, A., Bagyan, I., Heidrich, G., Lentzen, G., Seitz, H., Rampp, M., Schuster, S.C., Klenk, H.P., Pfeiffer, F. and Oesterhelt, D., 2011. A blueprint of ectoine metabolism from the genome of the industrial producer Halomonas elongata DSM 2581T. Environmental microbiology, 13(8), pp.1973-1994. 33. Severin, J., Wohlfarth, A. and Galinski, E.A., 1992. The predominant role of recently discovered tetrahydropyrimidines for the osmoadaptation of halophilic eubacteria. Microbiology, 138(8), pp.1629-1638. 34. Spencer, J.P., Jenner, A., Aruoma, O.I., Evans, P.J., Kaur, H., Dexter, D.T., Jenner, P., Lees, A.J., Marsden, D.C. and Halliwell, B., 1994. Intense oxidative DNA damage promoted by l-DOPA and its metabolites implications for neurodegenerative disease. FEBS letters, 353(3), pp.246-250. 35. Tian, B., Sun, Z., Shen, S., Wang, H., Jiao, J., Wang, L., Hu, Y. and Hua, Y., 2009. Effects of carotenoids from Deinococcus radiodurans on protein oxidation. Letters in applied microbiology, 49(6), pp.689-694. 36. Ventosa, A. and Nieto, J.J., 1995. Biotechnological applications and potentialities of halophilic microorganisms. World Journal of Microbiology and Biotechnology, 11(1), pp.85- 94. 37. Ventosa, A., Nieto, J.J. and Oren, A., 1998. Biology of moderately halophilic aerobic bacteria. Microbiology and molecular biology reviews, 62(2), pp.504-544. 38. Wang, C., Zhu, D. and Nagata, S., 2006. Supplementation effects of hydroxyectoine on proline uptake of downshocked Brevibacterium sp. JCM 6894. Journal of bioscience and bioengineering, 101(2), pp.178-184. 39. Xu, J., Chen, S. and Hu, Q., 2005. Antioxidant activity of brown pigment and extracts from black sesame seed (Sesamum indicum L.). Food chemistry, 91(1), pp.79-83.

Page 225

40. Yang, H.L., Chen, S.C., Chang, N.W., Chang, J.M., Lee, M.L., Tsai, P.C., Fu, H.H., Kao, W.W., Chiang, H.C., Wang, H.H. and Hseu, Y.C., 2006. Protection from oxidative damage using Bidens pilosa extracts in normal human erythrocytes. Food and Chemical Toxicology, 44(9), pp.1513-1521. 41. Zhang, L., Wang, Y., Zhang, C., Wang, Y., Zhu, D., Wang, C. and Nagata, S., 2006. Supplementation effect of ectoine on of phytase. Journal of bioscience and bioengineering, 102(6), pp.560-563.

Page 226