A Lakeside Tale
by Christoph
How small are bacteria ? And how big can they get as single cells ? For the lower end of the size scale, we know since the work of Jill Banfield's team that a multitude of ultra-small bacteria exist with a spherical diameters of ~0.25 µm and calculated cell volumes of ~0.009 µm3 (these 'Lilliputians' were featured here in STC). Much larger are newborn cells of Escherichia coli with a length of roughly 2 µm without flagella, and a diameter of approximately 1 µm. The trivial reason for these annoyingly vague numbers is that there is no fixed 'normal' size for E. coli. Cell volumes and, consequently, dry mass vary greatly with growth conditions, as much as by a factor of 5 between cells growing with a doubling time (Td ) of 100 min and those with a Td of 24 min; the slower growing ones also being smaller, of course.
The 'idealized' 2 µm x 1 µm E. coli cells, the fast growers, have the shape of sphero-cylindrical rods with a volume of 5π/12 µm3, that is ~1 µm3, which would account for a wet weight of ~1 pg (=10–12 g) assuming that they are just water. Proteins and other cell components have a slightly higher density than water, thus the weight of ~1 pg for an E. coli cell is, again, not precise but... easy to memorize. Most known bacteria have cell volumes that deviate at most by a factor of 5 from the ~1 µm3 E. coli volume. This for a good reason: unlike eukaryotic cells with their sophisticated intracellular transport systems, the cells of prokaryotes rely almost entirely on diffusion for cytoplasmic and trans-membrane transport of metabolites and, no less important, their protein 'tool kit' and plethora of RNAs. However, 'free' diffusion of molecules in the bacterial cytoplasm is restricted by macromolecular crowding, and the "1 – 5x E. coli volume" is apparently a viable compromise for cells with a single genome (there are exceptions, as always).
Figure 1. Dividing cell of A. oxaliferum. Stacked Overlay of bright-field images showing calcite crystals (CaCO3) and sulfur droplets (S0) with fluorescence microscopic images of the same cell stained for nucleic acids with SybrGreen I. Note that sulfur and nucleic acids spots are present in the grooves around the calcite bodies, but not at the same positions. In this cell, 244 DNA spots were counted using the software tool 'CountThem'. Both bright field and fluorescence images were taken as focus stacks of 22 images covering the full-cell depth and processed by the stacking program 'PICOLAY'. Source
And yet, there are a number of XXL-bacteria, so large that single cells of them are visible to the naked eye, and not just long filaments (=chains) of cells that we know, such as the Cyanobacteria (not to mention the up to 750 µm (!) long multinucleated cell of an E. coli metK mutant that strictly refuses to divide when starved for S‑adenosylmethionine ). Take Epulopiscium fishelsoni, a relative of the Clostridia (Gram-positive) and a gut symbiont of the brown surgeonfish (Acanthurus nigrofuscus), whose highly size-variable cells can reach widths of 80 µm and lengths of ~ 550 µm, accounting for volumes of ~350,000 µm³, or 5 orders of magnitude larger than that of the 'idealized' E. coli cell (featured by Elio here in STC ). Or take Thiomargarita namibiensis, a Gammaproteobacterium (Gram-negative) of the order Thiotrichales, whose globular cells have diameters of 100 – 300 µm (but can reach a diameter of 750 µm) and a volume 6 orders of magnitude larger than that of the 'idealized' E. coli cell (featured by Merry here in STC ). Also among the XXL bacteria are the disc-shaped cells of Beggiatoa with diameters of up to 160 µm, which when left undisturbed, can form microbial mats up to 17 m long crocheted from long filaments, or several distinct Thioploca species and their relative Thiolava veneris, and Marithrix (as you will realize from the links in this paragraph, we at STC are big fans of the XXL bacteria! ). Thiomargarita and the latter species belong to one branch of the order Thiotrichales within the Gammas, all share a chemolithotrophic metabolism and are capable of sulfur oxidation. All have large vacuoles that contain sulfur droplets and that, in the case of Thiomargarita, reduce the volume of the actual cellular cytoplasm in the globular cells by up to 90%. This is apparently not sufficient to achieve the 'viable compromise' for the cytoplasmic volume per genome (mentioned above ). Although genome analyses for these species are not yet complete, there is evidence that they all contain up to several hundred copies of their genome. As Mendell et al. wrote: "In large bacteria, genomic copies arrayed around the cellular periphery would permit transcription of any gene at disparate locations within the cell, thus reducing transit time of proteins and metabolites from site of synthesis or entry to their site of action. In this way, a large bacterium could function like a microcolony, with different regions of the cell independently responding to local stimuli, which would alleviate some of the pressure to remain small for the sake of rapid intracellular diffusive transport." Indeed, in this picture (Source), the DAPI-stained multiple chromosomes of a Thiomargarita cell appear aligned along the membrane like the Baily's beads in one of the photographs of the recent solar eclipse.
Enter Achromatium oxaliferum...
Figure 2. Drawings of Achromatium oxaliferum by W. Schewiakoff. The ovoid cells (1) contain large inclusions (J) that he thought were calcium oxalate, surrounded by a membrane, and distributed in the interior (2) of a layer that had a honey comb-like network structure ®. He supposed that this layer lies beneath a thick membrane or sheath (M). Along the nodes and connections of the comb-like structure, he saw numerous small inclusions that stained with hematoxylin and are, therefore, probably chromatin nodules (rK). Adapted from V. Salman, Thesis, and (1).
A. oxaliferum (Thiotrichales) is a 'newcomer' to this blog but not to microbiology (Fig. 1). According to Bergey's Manual (2015 Edition) Achromatium cells are "spherical to ovoid or cylindrical unicells with hemispherical ends. ... Cells vary in size from spheres of about 10 µm in diameter to giant cylindrical forms up to 30 × 125 µm (extremes are dividing stages); cells are on average 15 × 50 μm. ... Division of cells by binary constriction. Gram negative. Calcium carbonate [calcite] is deposited intracellularly. Additionally, the cells contain globules of S0 [elemental sulfur, oxidation state = 0]". This species was first described in 1893 by the Russian biologist Wladimir Schewiakoff (Владимир Шевяков, 1859 – 1930), who worked mostly on protists (Fig. 2). He was the first to observe the motility of Achromatium on microscope slides and described it as "rotary or jerking" (1). Schewiakoff did not see the intracellular sulfur globules, which can be seen dispersed in the cytoplasm in Figure 1 well apart from the nucleoids, and which are, obviously, not contained within a larger vacuole as in other Thiotrichales. He did, however, observe the nucleoids distributed in the cytoplasmic volume not occupied by the many round-shaped inclusions of amorphous calcite. He falsely took the latter for calcium oxalate, thus the name oxaliferum (whether the calcite bodies reside within the cytoplasm or in invaginations of the periplasm is not known yet). Achromatium has since been found worldwide, in freshwater and in the brackish water sediments.
In their recent study, Hans-Peter Grossart and his coworkers from the Leibniz Institute for Freshwater Ecology and Inland Fisheries (IGB), Berlin, describe surprising findings when trying to elucidate the genome of Achromatium oxaliferum from Lake Stechlin, NE Germany, which they did by DNA sequencing of 27 single and ~10,000 pooled "handcked" cells (2). Detailed analyses of the complete and partial sequences for 16S rRNA from single and from pooled cells showed that >99% were clearly related to Achromatium, excluding above-background contamination of their samples with other bacteria. The sequences had near identity in selected universally conserved regions but a considerable diversity in the hypervariable regions (they could thus exclude DNA sequencing flaws as a reason for the observed sequence heterogeneity ). The surprising heterogeneity among the 16S rRNA hypervariable regions was in the range one would readily accept for samples comprising various members of a taxonomic family or genus, not for a species (reminder: taxonomy classifies, in descending order, phylum > class > order > family > genus > species ). And this heterogeneity was not only found in their 10,000-cell pool but also among the single cell sequences! FISH analyses on single cells with a set of rRNA probes confirmed that different Achromatium rRNA sequences originate from intracellular diversity. Although rare, heterogeneity among rRNA sequences from a single species is not unprecedented, yet not to the extent found for Achromatium oxaliferum. Which, of course, made the analysis of other genes both necessary and thrilling.
Figure 3. Gene synteny in the tryptophan synthase operon (trpBA) region in A. oxaliferum. Bin = metagenomic sequences; Cell = single cell sequences; WMS3 = contig from isolate A. oxaliferum isolate WMS3; A. palustre = contig from the "Candidatus Achromatium palustre" genome sequence [LFCU00000000.1]. Square brackets demark contig borders. Source
They checked 131 markers whose genes are in most cases present as "single copy" in bacterial genomes. For the majority of them they found 15 – 20 sequence variants. Once again, a situation one would readily accept for samples comprising various members of a taxonomic family or genus, but not for a species. All these single-cell genomes contain multiple and different copies of some of the "single copy" genes, further supporting that we are dealing here with great intracellular diversity. Finally, they analyzed the synteny, that is, the gene context around selected single genes and several operons. Figure 3 shows their alignment of the trpBA region variants in their complete set of sequence contigs. It is immediately apparent that conservation of gene context is poor and in many cases the trpBA genes, the majority truncated for trpA, are flanked by transposase genes – transposons are unusually frequent in their sequences – and other easily movable genes, for example, R/M genes (a whole bunch of different R/M and methylase gene comprise the main difference among the ~200 sequenced Helicobacter pylori (Epsilonproteobacteria) strains ). Taken together, this diversity of alleles of bona fide "single copy" genes in an equally diverse gene context explains why the researchers failed to assemble a single complete (probably ) circular chromosome despite their collection of sequence contigs being sufficient give at least 2000‑fold coverage of a large prokaryotic genome of, say, 7 Mbp.
"Community-like genome in single cells..."
Ionescu et al. propose a model to account for their findings (Fig. 4). In short, an ancestral Achromatium found a way to multiply its chromosome to cope with its increased cell volume, much like its Thiotrichales siblings. This ancestor was, at one point, 'flooded' with transposons via phage infections or HGT. The quasi‑compartmentalization of the Achromatium cell by its calcite inclusions led to various integration events of the transposons into different chromosomes and subsequent transposon-triggered re-arrangements by recombination affecting primarily the 'local' chromosomes (intracellular genetic transfer (iGT) in contrast to HGT ). Gene conversion – the mechanism usually operative to 'streamline' identical chromosomes in a cell – does not occur on a cellular level in Achromatium but maintains genome stability at a local scale, thus fixing multiple versions of the same gene in different cellular locations. As a consequence, the genomes of daughter cells differ from each other and also from that of their mother cell.
Figure 4. Proposed model for generation of large intracellular and subsequent population genetic diversity. a Genome replication in an ancestral Achromatium cell a is followed by (b) the acquisition of an unusually high number of transposases via undetermined mechanisms (phages, HGT). c The genomes in the polyploid cell undergo a process of intracellular gene transfer (iGT) with limited transfer between cell parts due to the multiple calcite bodies which can be seen in the insert. The new chimeric genomes further interact increasing the cellular diversity while maintaining conserved regions such as operons intact. d Overall, this results in a cell with multiple different chromosomes represented by the different colors of the chromosomes. Gene conversion does not occur on a cellular level but maintains genome stability at a local scale and thus fixing multiple version of the same gene in different cellular locations. e A dividing cell recruits part of the genomic material generating two daughter cells different from each other as well as from the mother cell. Scale bar in SEM insert is 10 μm. Source
The authors literally hide their unprecedented finding of multiple different chromosomes in one single cell by casually talking of "polyploidy" in this organism. Polyploidy is a useful term for the description of eukaryotic genomes when it comes to counting the numbers of (nearly) identical chromosomes in a nucleus (think of the various di‑, tetra‑, and hexaploid varieties of wheat, or the different ploidy of developing and adult human heart muscle cells ). For bacteria, the term "polynucleoidy" would be more appropriate but sounds outright gross (try to say it loud without knotting your tongue! Elio discussed this years ago here in STC ). Therefore, "bacterial polyploidy" should be taken as a proxy for the situation in Beggiatoa, Thiomargarita, and Epulopiscium where multiple (almost) identical chromosomes are maintained within one cell: it's more of a copy-number issue, sort of. Not so in Achromatium, which presents a "Community-like genome in single cells..." (title of the Ionescu et al. paper ).
The sequence data suggest that the genes of this 'community genome' are phylogenetically related on the (taxonomic) genus level, and distributed over a cohort of ~200 chromosomes within single Achromatium cells. If this in not polyploidy – and it is not! – bacteriology has a novel "drawer". Yet, before putting an appropriate label on this "drawer" a confirmation of the results by a different experimental approach would be desirable – in keeping with Pierre-Simon de Laplace's (1749 – 1827) demand that "The weight of evidence for an extraordinary claim must be proportioned to its strangeness". One could, for example, dive deeper into the sizes and gene content of single Achromatium chromosomes by applying Pulsed-Field Gel Electrophoresis (PFGE) for their separation, in combination with subsequent Southern Hybridization experiments and sequencing. It goes without saying that one crucial question that Grossart and his coworkers are eager to tackle is the mode of distribution of the different chromosomes to daughter cells during cell division in a manner that maintains the integrity of Achromatium's 'community genome' in the progeny and thus its character as a distinct species.
A Rendezvous
Figure 5. Jetty at the shore of Lake Stechlin, NE Germany (53°08'30.0"N 13°01'46.2"E). 2017 CC BY-NC Christoph Weigel
So far, A. oxaliferum has not been grown in the lab. But the researchers are lucky since their lab is located directly at the shore of Lake Stechlin (Fig. 5): whenever they need cells for experiments – a daily routine, as was my impression during a visit – they take fresh samples from the lake sediment where the bacterium thrives in high numbers. As Hans-Peter Grossart told me:"...the sediment surface is oxic for ca. 0.5 cm deep into the sediment. Achromatium is sitting exactly in the redox gradient where the concentrations of H2S and O2 meet. That is in the upper centimeter or a bit deeper. We just take cores and look at the distribution of Achromatium. Most of the cells are definitively in the upper 1 – 2 cm. However, since it is a large sulfur bacterium it needs H2S to some extent and tries to avoid too high oxygen concentrations". Achromatium cells are quite robust, large, and rather heavy due to their calcite inclusions. Therefore, they can be 'washed' by resuspension of the 'right' sections of sample cores: coarse sand particles fall down immediately while it takes Achromatium cells a little longer to sediment. Thus, the separation of Achromatium from other floating bacteria and amoebae is no big deal. I guess it was this sedimentation behavior that led the researchers to speculate how Achromatium can actively search for its physiological 'Goldilocks zone' in the redox gradient: while cells can 'climb' towards higher O2 concentrations through the porous sandy sediment due to their known ability for gliding motion, the calcite inclusions, in turn, allow them to sink towards higher H2S concentrations if necessary. As I was told, experiments to test this hypothesis are underway.
Figure 6. Schewiakoff's drawing of a dividing Achromatium cell. Adatpted from (1)
When I was allowed to peek at 'freshly washed' Achromatium cells through a microscope, notably at just 6x magnification, I saw a few Euglena flagellates buzzing around, some other tiny unicellular euks, and several dozens of Achromatium cells, somewhat thicker than the translucent Euglena but ~1/3 their length, and easily recognizable by their white, potato-shaped inclusions of amorphous calcite (which rapidly morph into the typically rhomboid calcite crystals when released from the cells, as Heribert Cypionka, one of the authors, told me ). The cells were apparently doing well since many were busy with cell division in a very regular manner, that is, constricting at midcell (as Schewiakoff had already observed 1.25 centuries ago, see Fig. 6 ). I could have easily picked single cells – as they used for DNA sequencing – from the suspension with a micropipette. When I turned away from the ocular and inspected the Petri dish with the Achromatium cells I was still able to see them but would have failed to count their calcite "potatoes." For a microbiologist trained to observe his 'objects of desire' under the microscope at >400x magnification, it was, admittedly, a stunning experience to actually see individual bacteria with the naked eye (ok, equipped with eyeglasses in my case ).
References
(1) Schewiakoff W. 1897. Ueber einen neuen bacterienähnlichen Organismus des Süsswassers. Verhandlungen des Naturhistorisch-medizinischen Vereins zu Heidelberg, 5, 44 – 79. (Open Access PDF here)
(2) Ionescu D, Bizic-Ionescu M, De Maio N, Cypionka H, Grossart HP. 2017. Community-like genome in single cells of the sulfur bacterium Achromatium oxaliferum. Nat Commun, 8 (1), 455. PMID 22230956 (Open Access PDF here)
