Department of Microbiology, Radboud University, 6525 HP Nijmegen, NETHERLANDS.
Bacteria perform a variety of complex metabolic processes which are of global importance in nutrient cycling and primary production in aquatic environments. In these environments, bacteria are often associated with surfaces where they form multispecies assemblages which are called biofilms. While these assemblages can form on basically every surface, bacteria associated with aquatic eukaryotic phototrophs, such as macroalgae or seagrass, were found to play important roles in supporting the growth of their hosts while they -at the same time- live off the nutrients provided by the host organism. Major bacterial groups found in these associations are for example Proteobacteria or Bacteroidetes, but recently the abundance and importance of the phyla Planctomycetes and Verrucomicrobia has become evident. Planctomycetes are bacteria with a variety of seemingly unusual features and genome analyses provide evidence for a role in bioactive molecule production by this bacterial group. However, the planctomycetal phylogeny is still scarce, with many described genera being represented by only a single species. Thus, the aims of the research presented in this thesis were the cultivation, description and in depth analysis of novel species of Planctomycetes and Verrucomicrobia. In addition, elucidation of the bacterial community composition of macroalgal (Laminaria sp., Fucus sp., Ulva sp.) and seagrass (Posidonia oceanica biofilms by next generation sequencing and different microscopic techniques was performed. Also, attachment of Planctomycetes to algal particles was investigated.
Chapter 1 starts by giving an overview and a general introduction on Planctomycetes and Verrucomicrobia in aquatic habitats. Chapter 2 describes the analysis of the epiphytic biofilm bacterial communities of three species of North Sea macroalgae by next generation sequencing. In addition, the biofilm morphology was investigated by scanning electron microscopy and sample material was used for the selective cultivation of novel Planctomycetes species. Five novel strains were brought into pure culture and described as four novel genera and one novel species. Microscopical investigations of these strains revealed a set of interesting morphological features, while genome sequencing and genome analysis hinted the existence of several putative gene clusters related to secondary metabolite production. Also, attachment of the novel strains to macroalgal particles was investigated and morphological changes observed in response to the algal particles were monitored.
In chapter 3, the biofilm bacterial community of young and aged P. oceanica leafs was investigated for the first time with next generation sequencing and Planctomycetes were detected as dominant fraction of the bacterial community. In addition, two novel planctomycetal strains, representing two new planctomycetal genera, were isolated and thoroughly described. The genomes of both strains were sequenced and several gene clusters with possible relevance in secondary metabolite production were detected. In response to changing temperature, one strain showed significant changes in its growth pattern, switching from aggregated to homogenous growth. However, the mechanism underlying this growth pattern switch, at this point, remains elusive.
In chapter 4, three novel bacterial species belonging to the subdivision 4 of the phylum Verrucomicrobia were isolated from a freshwater pond and brought into pure culture. All strains belong to a new verrucomicrobial genus which is given the name Lacunisphaera. For the subdivision 4 of the Verrucomicrobia, state-of-knowledge is that its members lack the otherwise universal bacterial cell wall component peptidoglycan (PG) and thus pose as an exception among free-living bacteria. The presence of peptidoglycan was investigated by extracting cell sacculi of one novel strain, L. limnophila IG16bT, while at the same time, whole cell hydrolysates of all three novel strains were investigated and the PG marker molecules diaminopimelic acid and surprisingly, ornithine, were detected. Furthermore, bioinformatic methods as well as enzymatic treatment- and antibiotic resistance experiments were performed and support the claim that peptidoglycan is present in the newly described strains and related type strains of the subdivision 4 Verrucomicrobia.
Conclusively, chapter 5 revisits the results and data obtained in the previous chapters and discusses conceivable subsequent research topics based on the findings this thesis presented.