Microbial Diversity of Soda Lake Habitats Von der Gemeinsamen Naturwissenschaftlichen Fakultät der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigte D i s s e r t a t i o n von Susanne Baumgarte aus Fritzlar 1. Referent: Prof. Dr. K. N. Timmis 2. Referent: Prof. Dr. E. Stackebrandt eingereicht am: 26.08.2002 mündliche Prüfung (Disputation) am: 10.01.2003 2003 Vorveröffentlichungen der Dissertation Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Gemeinsamen Naturwissenschaftlichen Fakultät, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen vorab veröffentlicht: Publikationen Baumgarte, S., Moore, E. R. & Tindall, B. J. (2001). Re-examining the 16S rDNA sequence of Halomonas salina. International Journal of Systematic and Evolutionary Microbiology 51: 51-53. Tagungsbeiträge Baumgarte, S., Mau, M., Bennasar, A., Moore, E. R., Tindall, B. J. & Timmis, K. N. (1999). Archaeal diversity in soda lake habitats. (Vortrag). Jahrestagung der VAAM, Göttingen. Baumgarte, S., Tindall, B. J., Mau, M., Bennasar, A., Timmis, K. N. & Moore, E. R. (1998). Bacterial and archaeal diversity in an African soda lake. (Poster). Körber Symposium on Molecular and Microsensor Studies of Microbial Communities, Bremen. II Contents 1. Introduction............................................................................................................... 1 1.1. The soda lake environment .................................................................................. 1 1.2. Microbial diversity of soda lakes ......................................................................... 8 1.3. Ribosomal RNA(-genes) and molecular microbial ecology.................................16 1.4. Aim of the project...............................................................................................23 2. Materials and methods.............................................................................................24 2.1. Samples........ ......................................................................................................24 2.2. Extraction of DNA..............................................................................................25 2.2.1. Preparation of genomic DNA from pure cultures of bacteria .......................25 2.2.2. Rapid preparation of genomic DNA from bacterial colonies........................25 2.2.3. Extraction of DNA from sediment samples..................................................26 2.2.4. Determination of DNA concentration ..........................................................27 2.3. Polymerase chain reaction (PCR)........................................................................27 2.3.1. Purification of PCR products.......................................................................28 2.3.2. Oligonucleotide primers for PCR ................................................................29 2.4. Cloning of amplified 16S rDNA .........................................................................30 2.4.1. Blue/white screening for recombinant plasmids...........................................30 2.4.2. Size screening for recombinant plasmids.....................................................30 2.4.3. Storage of clones .........................................................................................31 2.4.4. Preparation of plasmid DNA .......................................................................31 2.5. 16S rDNA sequencing ........................................................................................31 2.5.1. Thermocycler protocol ................................................................................32 2.5.2. Purification of extension products ...............................................................32 III 2.5.3. Analysis of sequence data............................................................................32 2.6. DNA hybridisation..............................................................................................32 2.6.1. Dot blotting of DNA....................................................................................32 2.6.2. Dot blot pre-hybridisation and hybridisation ...............................................33 2.6.3. Stringency washing .....................................................................................33 2.6.4. Chemiluminescense detection......................................................................34 2.6.5. Rehybridisation ...........................................................................................34 2.6.6. Oligonucleotide probes................................................................................34 2.7. Amplified ribosomal DNA restriction analysis (ARDRA) ..................................34 3. Results and discussion..............................................................................................36 3.1. Screening of 16S rDNA molecules .....................................................................36 3.1.1. DNA sequencing .........................................................................................36 3.1.2. Fingerprinting analysis ................................................................................36 3.1.3. Non-radioactive colony hybridisation..........................................................38 3.2. Cultivation-independent analysis of microbial diversity .....................................42 3.2.1. Extraction of DNA ......................................................................................42 3.2.2. PCR amplification .......................................................................................44 3.2.3. Cloning .......................................................................................................45 3.2.3.1. Generation of 16S rDNA clone libraries from serial-diluted DNA ....................................................................................................45 3.2.3.2. Regeneration of a ligation-independent cloning (LIC) vector...............46 3.2.4. Analysis of the predominant bacterial 16S rDNA sequence types................55 3.2.4.1. Predominance of sequence types of the Cyanobacteria ........................57 3.2.4.2. Sequence types related to the Firmicutes..............................................65 3.2.4.2.1. Sequence types related to the Bacilli .............................................. 65 3.2.4.2.2. Sequence types related to the Clostridia......................................... 70 3.2.4.3. Sequence types related to the Proteobacteria.......................................78 IV 3.2.4.3.1. Sequence types related to the Alpha-Proteobacteria....................... 78 3.2.4.3.2. Sequence types related to the Gamma-Proteobacteria.................... 79 3.2.4.3.3. Sequence types related to the Delta-Proteobacteria ....................... 85 3.2.4.4. Sequence types related to the Bacteroidetes.........................................85 3.2.5. Analysis of the “overall” diversity of bacterial 16S rDNA sequences..........87 3.2.5.1. Non-radioactive colony and dot-blot hybridisations .............................87 3.2.5.2. ARDRA fingerprinting.........................................................................88 3.2.5.3. 16S rDNA sequence determination.......................................................89 3.2.5.3.1. Sequence types related to the Bacilli .............................................. 92 3.2.5.3.2. Sequence types related to the Clostridia......................................... 93 3.2.5.3.3. Sequence types related to the Alpha-Proteobacteria....................... 98 3.2.5.3.4. Sequence types related to the Gamma-Proteobacteria.................... 99 3.2.5.3.5. Sequence types related to the Delta-Proteobacteria ..................... 101 3.2.5.3.6. Sequence types related to the Bacteroidetes ................................. 102 3.2.5.3.7. Chimeric sequences ...................................................................... 102 3.2.6. Analysis of the archaeal 16S rDNA clone library ......................................106 3.2.6.1. ARDRA fingerprinting.......................................................................106 3.2.6.2. 16S rDNA sequence determination.....................................................107 3.2.7. Statistical approaches to estimating 16S rDNA sequence diversity............116 3.3. Cultivation-dependent analysis of microbial diversity ......................................122 3.3.1. Analysis of Archaea isolates derived from other haloalkaline environments .............................................................................................122 3.3.1.1. Halobacteria.......................................................................................122 3.3.1.2. ARDRA fingerprinting.......................................................................125 3.3.1.3. 16S rDNA sequence determination.....................................................129 3.3.2. Analysis of aerobic sporeformers isolated from soda lake samples............136 3.3.2.1. ARDRA fingerprinting.......................................................................136 3.3.2.2. 16S rDNA sequence determination.....................................................137 3.3.3. Analysis of a unicellular cyanobacterium isolated from Lake Magadi .......141 3.3.3.1. Generation of a 16S rDNA clone library ............................................142 V 3.3.3.2. Screening of transformants by using colony hybridisation