Achromatium is the largest freshwater bacterium known to date. It was first described in the 19th century, and until recently it was believed to occur exclusively in freshwater environments. Like other large sulfur bacteria, Achromatium contains a high number of chromosomes (polyploidy). In our study, we counted up to 300 DNA spots per cell, yet it remains unclear whether each of the spots contains one or more chromosomes. What makes Achromatium so special among the microbial realm is the large number of calcite crystals per cell whose ecological purpose still remains elusive (Fig. 1).
Fig. 1 A dividing Achromatium cell containing large calcite crystals and elemental sulfur globules. DNA staining (green) highlights the polyploidic nature of this bacterium.
Achromatium lives in coastal sediments of lakes or estuaries located at the oxic-anoxic boundary. Yet, it is not available in culture, and thus our goal was to use metagnomics to get a better understanding on its physiology and ecological role in the environment.
For this, we isolated and cleaned roughly 10,000 cells from a few grams of sediment from the shore of Lake Stechlin, Germany, and sequenced their metagenome. Thanks to our cleaning step the metagenome was almost free of non-Achromatium sequences even though these cells are typically covered with ectosymbionts. Nevertheless, it appeared that it consisted of an Achromatium community made of multiple genera and species. The Achromatium rRNA obtained from the metagenome suggested the presence of several clades spreading widely across the known Achromatium sequences.
However, copies of protein-coding genes were as distant as if coming from different bacterial families rather than the same species. Gene synteny in operons was not always conserved, and a large number of transposases could be identified. We used FISH to check whether these are different species or the same organisms. We could show that most cells lit up with two or three different oligonucleotide probes, whereby the signals did not entirely overlap (Fig. 2).
Fig. 2 Fluorescence in-situ hybridization image of a DAPI stained Achromatium cell (A) labeled with probes targeting 3 different 16S rRNA sequence clusters (B-D) and the matching superimposed images (E-H).
Therefore, the following questions arose: Could single cells harbor so much diversity? Could genes hop on and off different chromosomes and diverge at the same time? To validate these questions and combat obvious skepticism related to such far reaching ideas, we decided to sequence a fragment of the 16S rRNA as well as the whole genome from multiple single cells. The outcome was astonishing! The 16S diversity was extremely large even after applying the most stringent bioinformatics algorithms. Additionally, copies of the same protein coding genes were as distant in single cells as across our metagenomic data.
Based on our findings, we derived a hypothetical model that could explain this phenomenon relying on the physical separation of chromosome clusters inside each cell. Although our study well demonstrates the presence of the phenomenon, it remains unclear which mechanisms are responsible for this extensive diversity while allowing Achromatium to maintain its core characteristics.
Our future studies will address the most pressing questions: Can the cell regulate the expression of different version of the same gene? Are different genes expressed in different locations of the cell? Is Achromatium an example of a unique form of bacterial multicellularity? And last, is Achromatium unique among other polyploid microorganisms? From the answers to these questions we expect a deeper insight into the molecular mechanisms which may allow for the observed high genetic diversity through multiple but different copies of genomes in a single polyploidic bacterial cell. We propose that this form of polyploidy could be another, yet overseen, route to the emergence of multicellular organisms.
The paper in Nature Communications is here: http://go.nature.com/2eK4HiQ
For additional details:
Danny Ionescu: ionescu@igb-berlin.de
Hans-Peter Grossart: hgrossart@igb-berlin.de
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