Research Projects

Verena Carvalho and Alvin cores
Working with sediment cores.
Thiomargarita
Thiomargarita, the giant sulfur bacteria from Namibia.

Ecophysiology of the SAR202 Chloroflexi group

In the framework of the ABYSS project (Assessment of bacterial life and matter cycling in deep-sea surface sediments), funded by the European Research Council, we are studying the abundant, yet, uncharacterized group SAR202 in the phylum Chloroflexi. These bacteria occur in high numbers in ocean waters with increasing density towards the benthos. In deepsea sediments, they can make up a major portion of the local communities. Using molecular techniques, such as tag sequencing, single-cell genomics, and flurescence in situ hybridization, we are investigating the ecophysiological role of these bacteria.

 

Polyploidy and clonality in giant sulfur bacteria

A single cell of the giant sulfur bacteria (Gammaproteobacteria) can be up to several hundred micrometer in size. DNA staining with DAPI or SybrGreen reveals numerous hotspots of DNA across the enormous cell body - they are uncountable in number and vary in size. Another giant bacterium with a similar pattern of DNA distribution (Epulopiscium (Firmicutes); average cell size 50x300 µm) revealed hundreds of thousands of genome copies per cell, meaning it is highly polyploid (Mendell et al 2008). The selection of a few genes of Epulopiscium showed that copies were highly identical.
To study the clonality across the cells of a filamentous organism, as well as the genome plasticity with a polyploid cell we used the giant "Candidatus Marithrix sp." as a model organism. We manually separated sections of a single filament of the sulfur bacterium, and sequenced the genomes therein. The analysis showed highly identical genetic material across the entire filament. However, when making use of the high coverage/depth of the next generation sequencing technology the genome plasticity became apparent. Using the reads and comparing nucleotides per position within a filament segment high numbers of variabilities were observed. This heterogeneity per position was higher by at least an order of magnitude than when using the aligned (collapsed) contigs and comparing nucleotide positions across all (aligned) genomes of the filament. We conclude that among a few polyploid cells, and maybe already at the level of a single polyploid cell, this heterogeneity among the many genome copies might represent hotspots of DNA mutation and microevolution (Salman-Carvalho et al. 2016). Testing this hypothesis on a single polyploid cell is the next step of our research, as well as the enumeration of genome copies of a single cell of sulfur bacteria.

Thiomargarita DAPI staining
A single Thiomargarita cell stained with DAPI. Across the entire cell, numerous hotspots of DNA are visible, and the cell is noticeably polyploid, i.e. contains multiple genome copies. Image from Angert 2012 Annu. Rev. Microbiol. 66:197-212.

Ultrastructure in Achromatium

Achromatium also belong to the group of giant sulfur bacteria, although delineating into a different family and measuring "only" up to 100 µm in diameter. Besides being likewise polyploid as their cousins, these bacteria have a unique feature among all known Bacteria and Archaea. The cells possess multiple inclusions that contain colloidal calcite. These rock-like structures fill almost the entire body of the cell, leaving only very little space for the cytoplasm and other inclusions such as elemental sulfur. The localization of the different parts of their biomass - periplasm, cytoplasm, membranes, all the different types of inclusions- is subject of our current work. We use different techniques for visualization, i.e. dyes, light and fluorescence microscopy, electron microscopes, and raman spectroscopy.
We also investigate the specific ecophysiology of freshwater and marine populations of these interesting gradient organisms. With chemotaxis experiments and by collecting cells from different layers of the sediment we try to reveal their behavior to get insights into their niche adaptation. Naturally, we also aim at getting some implications on the biological role of the calcite inclusions (Salman et al 2015).

Achromatium
Achromatium cells contain multiple, light-refracting calcite inclusions. Despite these "rocks", the bacteria are motile by rolling, and occur in sulfidic freshwater and marine sediments. Image from Salman et al 2015 ISMEJ 9:, 2503-2514.

16S rRNA introns

The gene for the small ribosomal subunit, the 16S rRNA gene, is used as a modern chronometer to reveal phylogenetic relationships among all living organisms. The conserved structure (length, composition) is of uttermost importance to qualify for this purpose. All standard techniques that involve the specific amplification of this genetic region are designed according to its consistency.
We investigated the 16S rRNA genes of the giant sulfur bacteria of the family Beggiatoaceae and revealed that about 70% of the analysed single cells and filaments contained multiple introns in their 16S rRNA genes - elongating the gene up to ~3000 kbp. These introns were autonomous and could self-splice out of the precursor RNA, leaving the ribosomes of the host organism unharmed. This finding, though, has major implications for our current understanding of genomic conservation and the design of "universal" techniques to reveal the diversity of organsims in natural populations (Salman et al 2012).

group I intron
Secondary structure a group I intron identified in the 16S rRNA genes of giant sulfur bacteria. Salman et al 2012 PNAS 109: 4203–4208.

Field work / Expeditions

2014 Research Station Lizard Island, Great Barrier Reef, Australia
  RV Atlantis AT26-13 with submersible ALVIN, Gulf of Mexico, USA
  RV Mirabilis, Benguela Upwelling System, Namibia
2011 RV Maria S. Merian, Benguela Upwelling System, Namibia
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