Diversity studies and molecular analyses with single cells and filaments of large, colorless sulfur bacteria

Large sulfur bacteria feature conspicuous morphologies that are usually visible with the naked eye. Most representatives were already described in the 19th and early 20th century and it needed nearly another 100 years until a new morphotype of large sulfur bacteria was discovered. This discovery encouraged a search for other yet unknown types in this group of bacteria and indeed led to the finding of a new type in two marine seep settings. This novel morphotype is presented in Chapter 2 and is the first non-filamentous member in the family Beggiatoaceae that shows a dimorphic life cycle, exhibiting alternation between sessile and free-living forms. In Chapter 3, another three novel morphotypes are presented, which were discovered in Namibian sediments. The detection of these novel morphotypes of large sulfur bacteria led to the necessity for an improved phylogenetic classification of the entire group. Earlier attempts to sequence the 16S rRNA genes of the large sulfur bacteria resulted in only few nearly full-length sequences, whereas some genera were even represented by only partial sequences. Accordingly, differentiation of genera in this group is still based on morphological features. Now, Chapter 3 presents the sequencing of 16S rRNA genes and ITS regions of more than 100 individual cells and filaments of large sulfur bacteria, revealing a major insight into the phylogeny of these extraordinary bacteria. It is demonstrated that the traditional, morphology-based classification does not correlate with the phylogeny derived from 16S rRNA gene sequences. Consequently, a reclassification of the entire family is proposed, being completely independent from former morphological categories. Sequencing single cells and filaments of large sulfur bacteria furthermore revealed that they represent the first group of bacteria, which commonly contain large and numerous introns in their 16S rRNA genes. In Chapter 4, it is demonstrated that the introns are removed efficiently from the rRNA precursor, thereby ligating the two exons, and are not present in the native ribosome. Furthermore, it was experimentally verified that a commonly applied PCR approach introduces a length heterogeneity bias and systematically discriminates against enlarged genes and favors the amplification of shorter homologues. Consequently, this fact questions the universality of 16S rRNA-based clone libraries for diversity studies. At least the group of large sulfur bacteria is systematically discriminated against in such universal PCR approaches and it can be assumed that this PCR bias also affects a yet unknown amount of other microorganisms.