Access to Electronic Thesis Author: Khalid Salim Al-Abri Thesis title: USE OF MOLECULAR APPROACHES TO STUDY THE OCCURRENCE OF EXTREMOPHILES AND EXTREMODURES IN NON-EXTREME ENVIRONMENTS Qualification: PhD This electronic thesis is protected by the Copyright, Designs and Patents Act 1988. No reproduction is permitted without consent of the author. It is also protected by the Creative Commons Licence allowing Attributions-Non-commercial-No derivatives. If this electronic thesis has been edited by the author it will be indicated as such on the title page and in the text. USE OF MOLECULAR APPROACHES TO STUDY THE OCCURRENCE OF EXTREMOPHILES AND EXTREMODURES IN NON-EXTREME ENVIRONMENTS By Khalid Salim Al-Abri Msc., University of Sultan Qaboos, Muscat, Oman Mphil, University of Sheffield, England Thesis submitted in partial fulfillment for the requirements of the Degree of Doctor of Philosophy in the Department of Molecular Biology and Biotechnology, University of Sheffield, England 2011 Introductory Pages I DEDICATION To the memory of my father, loving mother, wife “Muneera” and son “Anas”, brothers and sisters. Introductory Pages II ACKNOWLEDGEMENTS Above all, I thank Allah for helping me in completing this project. I wish to express my thanks to my supervisor Professor Milton Wainwright, for his guidance, supervision, support, understanding and help in this project. In addition, he also stood beside me in all difficulties that faced me during study. My thanks are due to Dr. D. J. Gilmour for his co-supervision, technical assistance, his time and understanding that made some of my laboratory work easier. In the Ministry of Regional Municipalities and Water Resources, I am particularly grateful to Engineer Said Al Alawi, Director General of Health Control, for allowing me to carry out my PhD study at the University of Sheffield. My deepest and special thanks are due to my beloved mother and wife for their support, patience and continuous encouragement throughout the period of my study. Finally, I would like to extend my sincere thanks to colleagues in Wainwright’s laboratory (G10) for constant encouragement and support. Special thanks to my best friend in MBB department Suleiman Alnaimat who was accompanying me thought-out the period of my study as well as Miss Reda Amasha for her encouragement and support, mainly making “Du’a”. Introductory Pages III ABSTRACT A number of samples were collected from various extreme and non-extreme environments. A range of molecular approaches, in addition to classical microbiology techniques, were used to isolate microorganisms, mainly archaea and bacteria from unusual environments. Halo-bacteria and archaea from non-saline environments and alkaliphilic bacteria and archaea capable of withstanding low pH values were isolated and identified using 16S rRNA gene cloning, PCR amplification and phylogenetic analysis. Detailed analysis of their halophilic and alkaliphilic physiology for adaptation in the environment was investigated using nuclear magnetic resonance spectroscopy (NMR) to detect the compatible solutes and atomic emission spectophotometery (AES) to measure the cellular potassium ions. In addition, ultraviolet-type C tolerant bacteria were isolated from terrestrial environments using phylogenetic analysis of the 16S rRNA gene sequences. A selection of other molecular techniques were used in this thesis. Fluorescent in situ hybridisation (FISH) allowed simultaneous identification, enumeration and visualization of microbial cells of the two domains; Archaea and Bacteria within a community in their natural habitat. In addition, using EZ-PCR test, culturable- independent Mycoplasma DNA in environmental samples was detected. Further investigation, using other advanced molecular techniques, into the physiology of extremophilic microorganisms found in non-extreme environment may provide a better understanding of the microbial interactions and the essential roles which different species play in the environment. Introductory Pages IV SUMMARY Summary of Aims The aims of this study were to: a) Determine if halophilic and halotolerant bacteria as well as archaea can be isolated from non-saline environments and if so to identify them using molecular methods. b) Determine how halophilic and halotolerant bacteria and archaea, isolated from non-saline environments, can survive extreme saline conditions. c) Conduct studies on the screening and isolation of alkaliphilic and alkalitolerant bacteria and archaea which are also able to grow in acidic media. d) Conduct studies on the question-how do presumptive alkalitolerant and alkaliphiles survive in such alkaline as well as low pH conditions?- e) Conduct studies on the isolation of ultraviolet-type C tolerant bacteria from terrestrial environments using phylogenetic analysis of the 16S rRNA gene sequences. f) The FISH technique was also used to allocate the isolated microorganisms to either archaea or bacteria. g) Finally, studies were conducted to determine (using a PCR detecting method) if mycoplasmas can be isolated from extreme and non-extreme environments. The first aim of this study was to determine if halophilic and halotolerant bacteria as well as archaea could be isolated from non-saline environments and if so to identify them using molecular methods; the following results were obtained: 1. Some 96% of the non-saline soil samples contained halophilic bacteria capable of growing at 1.0M , while 54% grew at 2.0M NaCl and 16 % were able to grow at 3.0M NaCl; only 6% of the isolates however, grew at 4.0M NaCl. 2. Some 12% of isolates from saline environmental samples yielded microorganisms capable of growing at 4.0M NaCl. Whereas, all samples Introductory Pages V produced growth at salt concentrations up to 2.0M and 62% of them produced bacteria capable of growing at 3.0M NaCl. 3. None of the air samples or single rain sample yielded any salt tolerant isolates. Two bacteria capable of growing at 2.0M NaCl only, were however, isolated from a hailstone sample. 4. The number of colony forming units isolated from both non-saline and saline soil decreased with increasing concentrations of NaCl added to plates containing Plate Count Agar. 5. The majority of the isolates obtained in this study were representatives of previously isolated organisms. 6. The isolates obtained from non-saline soils were Bacillus species, and analysis of partial sequences of their 16S rRNA genes showed that some of them exhibited similarities higher than 99% with those of Bacillus subtillis, Paenibacillus ehimensis and 100 % with Bacillus weihenstephanensis. No bacteria other than species of the genus Bacillus were isolated from non-saline samples. 7. Two isolates were obtained from hailstones on media containing 2M NaCl, one was identified as Pantoea dispersa (99.12 similarity) and the other as Staphylococcus sciuri sciuri (99.94 similaritiy). Bacillus pumilus isolated from the stratosphere grew in up to 3M NaCl. 8. The data presented here show that Bacilli are dominant among halophilic bacteria in non-saline environments; halophilic bacteria, in general, are however, not restricted to this genus. 9. The two archaeal isolates obtained from desert sands and desert varnish collected from Oman, were mixed with bacterial colonies and it was not Introductory Pages VI possible to separate them even with the use of antibiotics and as a result it was impossible to obtain valid sequence data for the identification of these isolates. 10. All previously mentioned isolates were isolated from non-saline environments which would be expected to act as a source of halotolerant but not halophilic bacteria. On the other hand, a strain of bacteria isolated from saline soil, showed strict obligate halophilic characteristics as it was not capable of growing, or even surviving without salt; optimum growth occurring at 1.0M and 2.0M NaCl as well as 4M salt. The study also focused on the question of how halophilic and halotolerant bacteria and archaea, isolated from non-saline environments, can survive extreme saline conditions. The following results were obtained: 1. In most isolates, the results for intracellular glycine betaine levels above 1.0M NaCl showed large variability, a general trend towards an increase in glycine betaine level with increasing salt concentration was observed. 2. NMR analyses of compatible solutes in all halotolerant isolates showed the accumulation of glycine betaine is important for osmotic adaptation of this isolate when growing in the presence of high NaCl concentrations (except when grown in M9 medium). The fact that betaine was the main osmoprotectant present in most isolates depended on the composition of the medium (i.e. LB medium contains yeast extract) showing that the presence of betaine in the cells was definitely not synthesized by the cells (e. g. de novo) and it appears that betaine is absolutely required as an organic osmolyte for these isolates when grown under stressed conditions. 3. The compatible solutes reported in this study mostly correspond with those reported in the literature and the results show that the halophilic isolates usually accumulated amino acids and polyols and their derivatives such as, glycerol, Introductory Pages VII ectoine, glutamate, proline and lysine, as well as quaternary amines such as glycine betaine which have no net charge at physiological pH and do not disturb metabolic processes. 4. This study also demonstrated that glycine betaine and glutamate were the main compatible solutes which were accumulated at high concentration