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INTO THE MICROBIAL COLOSSUS: EXPLORING NEWLY FOUND GIANT BACTERIA


The bacteria forms one of the most metabolically diverse groups along with archaea but due to various factors such as encounter with nutrients, elimination of wastes and transport of biomolecules the size of bacteria is restricted to few micrometers typically ranging from 2-8 μm in length and 0.2-2 μm. But there are few exceptions called giant bacteria which have size in the millimeter range and can be seen without a microscope. Recently, scientists discovered a bacterium that seems to defy all the norms about bacteria. This bacterium is larger than any other known giant bacterium by approximately 50-fold, making it possible to see with the naked eye. It has massive polyploidy and shows compartmentalization of genetic material and ribosomes in membrane-bound organelles. This bacterium has been proposed to be referred to as Candidatus (Ca.) Thiomargarita magnifica.

Thiomargarita magnifica


Morphology of Thiomargarita magnifica

Ca. T. magnifica is a centimeter long, single bacterium. Thiomargarita species are sulfur oxidizing gammaproteobacteria and are known to display polyphenism. This bacterium forms smooth filaments with an average length of 9.72 ± 4.25 mm with some filaments reaching a length of 20 mm. The filament is confirmed to be of a continuous cell for nearly its entire length with no division septa and only a few apical buds were separated by closed constriction which are daughter cells. Also the central vacuole was continuous along the whole filament and accounted for 73.2 % of the total volume and this minimizes the growth limitation due to reliance of the chemical diffusion.

 Size of Thiomargarita magnifica as compared to other prokaryotes and certain eukaryotes

TEM revealed a number of vesicles which correspond to refractive sulfur granules. The cytoplasm also contains many electron dense membrane bound compartments which are referred as “blebs of cytoplasm”, or  “putative endobionts” which may contain dispersed genomic material as polyploidy is evident in many giant bacteria.

TEM image of the apical constriction of the cell with sulfur granules (S), vacuoles (v) and pepins (arrowheads)

4′,6-Diamidino-2-phenylindole (DAPI) staining revealed that DNA is concentrated in membrane bound compartments in the cells of Ca. T. magnifica. They are also found to have electron dense structures 10-20 nm in size similar to ribosomes. The presence of ribosomes was confirmed by fluorescence in situ hybridisation (FISH) with probes targeting ribosomal DNA sequences. This compartmentalization of genome and ribosomes is similar to cellular organization of DNA and ribosomes in eukaryotes. The membrane bound structure containing ribosomes is termed as pepins which contain numerous ribosomes that appear as small, electron dense granules present throughout the pepins.


Fluorescence labeling of membrane using FM 1-43X and DNA using DAPI suggests the compartmentation of DNA within a membrane 

TEM image of a pepin containing number of ribosomes

Ca. T. magnifica also shows localization of ATP synthases around pepins and and throughout the complex membrane network of the entire cytoplasm and absent on the outer cell envelope rather than on the cell envelope like in the case of most of the bacterial species which is one of the factors potentially constricts bacterial cells size due to surface to volume ratio required to meet the energy requirements to a theoretical cell size to 10−14 m3. This arrangement allows the bacteria to meet its energy demands to maintain its large size. The localization of protein biosynthesis throughout the whole cell was tested using bioorthogonal noncanonical amino acid tagging (BONCAT) and the results were consistent with the results obtained from FISH and TEM and suggested that the protein biosynthesis is localized in small, round shaped areas that are similar to pepins in size and localization. But not all pepins were labeled and the labeled hotspots were observed at the site of constriction in the apical parts suggesting high translational activity in these areas.  



Visualization of localization of ATP synthases in the cytoplasmic membrane and pepins by immunohistochemistry using Anti–ATP synthase antibodies

Visualization of localization of protein biosynthesis using BONCAT

Whereas the doubling times for most bacteria ranges in the range of minutes to hours, Ca. T. magnifica requires upto 2 weeks to produce daughter cells but it does not double its volume for binary fission as they reproduce by detachment of small portions of apex constricts from the mother cells. They are polyploid meaning that they have multiple genome copies which are dispersed throughout the cell and have the highest estimated number of genome copies within the single cell which supports the local metabolic need and overall cellular growth and also decrease the suppressive pressure on gene allowing intracellular replication, reassortment, and divergence leading to greater genetic diversity and supports high level of genome conservation. The genome analysis revealed that almost all genome assemblies were estimated to be 91.0-93.7 % complete with a total length of 11.5-12.2 Mb which is much larger than normal bacteria genome length of 4.21 Mb and contained up to 11788 genes which is more that the three times of normal gene count of 3935 genes in prokaryotes. It also revealed that large fraction of genes are involved in sulfur oxidation and carbon fixation which suggests chemoautotrophy and lacked nearly all genes involved in dissimilatory and assimilatory of nitrate reduction and denitrification which suggests that nitrate can solely used as an electron acceptor. Also up to 25.9 % of the genome encodes many nonribosomal peptide synthetases  and polyketide synthases suggesting the presence of numerous secondary metabolic pathways that may be involved in antibiotic or bioactive compound production.   


The genome analysis also revealed that it lacks many genes which encodes for proteins like FtsA, ZipA, FtsE, and FtsX  which forms the core components of Z ring assembly and its regulation which is a major component in cell division and in contrast genes coding for cytoskeletal proteins FtsZ which is part of dew (division and cell wall) operon and core component of Z operon and genes encoding for proteins involved in regulation of Z ring assembly  were highly conserved and late divisome proteins like peptidoglycan polymerases FtsI, FtsW and FtsL, FtsQ, FtsL, FtsB, FtsK were completely absent suggesting the lack of cell division genes and the presence of complete cell elongation genes which may be the reason for the formation of long filaments of the bacterium.


Model of subcellular organization of Thiomargarita magnifica

Laboratory observations of this bacterium revealed the detachment of apical buds and its dispersal into the environment indicating the dispersive stages of the developmental cycle. The presence of intermediate stages from small attached cells to large filaments with apical constrictions were also observed which resembles the dimorphic life cycle in which stalked parent cells produce free living daughter cells. Because of this asymmetrical division only small amount of genome copies from pepins were transmitted to the daughter cell called germline genomes and this type of developmental cycle may be developed for dispersion similar to fruiting bodies of myxobacteria and aerial hyphae of Streptomyces spp.


The discovery of Thiomargarita magnifica, marks a pivotal moment in the field of microbiology. This remarkable bacterium, found in deep-sea sediments, challenges our understanding of microbial size limits and adaptations to extreme environments by also expands our understanding of microbial diversity and underscores the vastness of unexplored realms within our own planet.


REFERENCES:

  1. Levin, P. A., & Angert, E. R. (2015). Small but Mighty: Cell Size and Bacteria. Cold Spring Harbor perspectives in biology, 7(7), a019216. https://doi.org/10.1101/cshperspect.a019216

  2. Jean-Marie Volland et al.,A centimeter-long bacterium with DNA contained in metabolically active, membrane-bound organelles.Science376,1453-1458(2022).DOI:10.1126/science.abb3634



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