Bacteria and archaea are the most numerous and diverse animals on the planet, as we all know. Because just a small number of them have been isolated in culture, we know relatively little about their biology. Giant bacteria are found in at least four phyla and have cellular sizes in the tens or hundreds of microns. Sulfur-oxidizing gammaproteobacteria Thiomargarita namibiensis has been discovered at depths of up to 750 metres (average size: 180 metres). The existence of such bacterial behemoths raises the prospect that there are still more macrobacteria to be discovered.
Researchers present a new sessile filamentous Thiomargarita species from a sulfidic maritime habitat that dwarfs all other known giant bacteria by around 50 times. Massive polyploidy and a dimorphic developmental cycle, in which genome copies are asymmetrically partitioned into apparent dispersive daughter cells, are revealed by our multi-faceted imaging techniques. We show, for the first time, that centimeter-long Thiomargarita filaments are individual cells with genetic material and ribosomes segregated into a novel sort of membrane-bound organelle. The genomes of five single cells were sequenced and analysed, revealing new information about cell division and cell elongation mechanisms. These distinct cellular characteristics presumably allow the organism to develop to an extraordinarily big size while avoiding some biophysical and bioenergetic growth constraints.They suggest the name Thiomargarita magnifica for this species in honour of its enormous size (referred to below as Ca. Thiomargaritamagnifica).
Ca. T. magnifica DNA is contained in a novel type of membrane-bound bacterial organelle
Large Sulfur Bacteria (LSB) create lengthy filaments that can reach several centimetres in length, however they are made up of thousands of individual cells that are little longer than 200 m long. In shallow tropical marine mangroves of Guadeloupe, Lesser Antilles, we discovered seasonal "bouquets" of centimeter-long white filamentous Thiomargarita cells clinging to buried leaves of Rhizophora mangle. Sulfur-oxidizing gammaproteobacteria, Thiomargarita spp., are notable for their morphological diversity and polyphenism. The filaments found in Guadeloupe have the same morphology as sessile Thiomargarita-like cells found in deep-sea methane seeps. They were stalk-like for the majority of their length and gradually constricted towards the apical end, generating buds.These filaments were smooth and free of epibiotic bacteria and extracellular mucus matrix, in contrast to relatives who reside buried in sediment. Only the most apical constrictions closed completely to form 1-4 rod-shaped distinct cells of 0.21 0.05 mm, and budding filaments had an average length of 9.72 4.25 mm. Some filaments were 20.00 mm in length, far longer than any previously recorded single-celled prokaryote.
To better understand Ca. T. magnifica cells, we used osmium tetroxide or the fluorescent dye FM 1-43x to highlight membranes, and we used Hard X-ray Tomography (HXT, n=4) and Confocal Laser Scanning Microscopy (CLSM, n=6) to visualise entire filaments in 3D, as well as filament sections (up to 850.6 m long) with Transmission Electron Microscopy (TEM, n=15). Surprisingly, all approaches consistently revealed that each filament was one continuous cell over nearly its entire length, including the minor constrictions around the apical pole, with no division septa. A closed constriction separated only the most apical few buds from the filament, indicating that these were daughter cells.
Ca. T. magnifica cells, like other Large Sulfur Bacteria (LSB), had a large central vacuole that ran the length of the filament and accounted for 73.2 7.5 percent (n=4) of total volume. The cytoplasm was 3.34 1.48 m thick and restricted to the cell's perimeter. As evidenced by Energy Dispersive X-ray Spectroscopy, TEM revealed multiple electron lucent vesicles with a diameter of 2.40 1.03 m, which corresponded to the refractile granules seen with bright-field microscopy and resembled sulphur granules. The cytoplasmic membrane was covered by a thick outer layer of the cell envelope. Similar to other LSB, the cytoplasm of Ca. T. magnifica appeared to be compartmentalised in the form of dense regions.including numerous membrane-bound particles with a diameter of 1.31 0.70 m. Because many big bacteria have polyploidy, we speculated that some of these dense areas inside the cytoplasm, which were separate from massive sulphur granules, would contain distributed genetic material.
Ca. T. magnifica DNA is contained in a novel type of membrane-bound bacterial organelle
While bacteria were once thought to be uncompartmentalized "bags of enzymes," recent research has revealed that they have organelles that perform functions as diverse as anaerobic ammonium oxidation, photosynthesis, and magnetic orientation. Although some evidence for a putative membrane-bound DNA compartment filling most of the cell's volume in a member of the Atribacteria has been reported, no bacteria or archaea have been found to conclusively partition their genetic material in the manner of eukaryotes. Surprisingly, DAPI staining revealed that DNA in Ca. T. magnifica cells was concentrated in membrane-bound granules rather than being distributed throughout the cytoplasm, as is typical in bacteria. These DNA-containing entities also had electron-dense structures ranging in size from 10 to 20 nm, which are similar to ribosomes.FISH using probes specific for Thiomargarita ribosomal RNA sequences indicated that ribosomes were present and concentrated in these membrane-bound structures, which were found throughout the cell, including the apical buds. This DNA and ribosome compartmentalization resembles genomic compartmentalization in eukaryotes and indicates an unique cellular structure in bacteria. We recommend calling this bacterial organelle a "pepin" after pips, the numerous tiny seeds found in fruits such as watermelon and kiwi.
A highly polyploid cell with a large genome
All previously documented gigantic bacteria are polyploid, which means that their cells contain a significant number of genome copies – tens to tens of thousands – scattered throughout the cell, enabling local molecular machineries and overall cellular growth. In some LSB, polyploidy has been demonstrated to reduce selective pressure on genes, allowing intracellular gene duplication, reassortment, and divergence, as well as resulting in extraordinary intracellular genetic diversity. On the other hand, it may allow homologous recombination to balance genome copies and support a high level of genome conservation. Ca. T. magnifica, like all bacterial giants, appeared to be polyploid, with an average of 36,880 7,956 genome copies per millimetre of filament based on counts of DAPI stained DNA clusters on three CLSM 3D datasets.This is the largest number of genome copies that a single cell has been estimated to have. Its size is a factor of a million times larger than that of other big bacteria.
We amplified, sequenced, and assembled the DNA of five individual cells collected from a single sunken leaf to genomically characterise Ca. T. magnifica. With an Average Nucleotide Identity (ANI) of more than 99.5 percent, all five draught genomes looked to be very similar to one another. Variant analysis of single-cell genome sequences revealed that the population is genomically homogeneous, similar to other polyploid bacteria. The assemblies were assessed to be 91.0 percent to 93.7 percent complete, with total sequence lengths ranging from 11.5 to 12.2 megabytes. This is twice the size of the only other Thiomargarita species that has been sequenced, Ca. T. nelsonii, and well above the average bacterial genome size of 4.21 1.77 Mb.The genome of Ca. T. magnifica filament 4 had 11,788 genes, which was more than three times the median gene count of prokaryotes. Ca. T. magnifica has a genome as large as the baker yeast S. cerevisiae (12.1 Mb) and more genes than the model fungus Aspergillus nidulans (9,500 genes) for comparison with eukaryotic organisms.
A substantial number of genes for sulphur oxidation and carbon fixation were discovered in the genome, indicating chemoautotrophy, which is consistent with our other findings for thioautotrophy. Ca. T. magnifica, like its sister lineage Ca. T. nelsonii, encoded a wide range of metabolic abilities, with one notable exception: it lacked nearly all genes involved in dissimilatory and assimilatory nitrate reduction, as well as denitrification, with the exception of Nar and Nap nitrate reductases. This indicates that nitrate can only function as an electron acceptor. The huge number of genes encoding secondary metabolism could explain the remarkable lack of epibiotic bacteria, considering its size. Biosynthetic gene clusters account for 25.9% of all sequences.The genome encodes a slew of modular Non-Ribosomal Peptide Synthetases (NRPSs) and Polyketide Synthetases (PKSs) systems, pointing to a slew of secondary metabolism pathways that may be used to make antibiotics or therapeutic compounds.
With an atypical complement of cell division and cell elongation genes, the Ca. T. magnifica genome held clues for its unusual cell morphology. Many genes encoding essential cell division proteins were missing, including FtsA, ZipA, and FtsE-FtsX, which are essential for Z ring construction and control. whereas genes encoding the cytoskeletal protein FtsZ, which is a fundamental component of the Z ring and is part of the well-conserved dcw ("division and cell wall") operon, and proteins ZapA, ZapB, and ZapD, which interact with FtsZ and regulate Z ring formation, were conserved. Even more remarkable, all Ca. Thiomargarita genomes lacked the entire set of genes that encode late divisome proteins, including peptidoglycan polymerases FtsI and FtsW, as well as FtsQ, FtsL, FtsB, and FtsK.The whole set of genes for cell elongation proteins, three of which -mreD, rodZ, and peptidoglycan transpeptidase mrdA - have experienced recent duplications, with both copies placed near to each other on the chromosome, was in stark contrast to the lack of cell division genes. It's possible that Ca. T. magnifica's unusually long filaments are the result of an increased number of cell elongation genes combined with a lack of key cell division genes.
Dimorphic developmental cycle of Ca. T. magnifica
The eventual detachment of the apical bud from the filament and release into the environment was observed in laboratory observations of live Ca. T. magnifica, indicating a dispersive stage of the developmental cycle. We saw dozens of cells at all phases of development, from the tiniest connected cells that looked like terminal segments that had just settled to the biggest filaments with apical constrictions. The aquatic single-celled model system Caulobacter crescentus, as well as the multicellular segmented filamentous bacteria, have dimorphic life cycles in which stalked "parent" cells spawn free-living "daughter" cells, albeit on a smaller scale. Only a small fraction of the genome copies – contained in pepins in the most apical bud – were passed to the daughter cell due to this asymmetrical division strategy.If terminal buds are daughter cells, a developmental cycle similar to the fruiting bodies of social myxobacteria or the aerial hyphae of Streptomyces spp. may have evolved to enhance dispersion. This apparent life cycle is also similar to that of Zoothamnium niveum, a sulfur-oxidizing giant ciliate symbiosis, presumably indicating convergent evolution of developmental cycles across domains.
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