Miraculous Microbes
by the STC team
Surprising as the world of microbes may be, some stories are so far beyond our expectations that they border on the miraculous. We share some of these "Believe it or Not" stories.
1. Teaching E. coli how to fill up with foreign vesicles
(modified from a previous post) What if we told you that engineering a single protein into E. coli is sufficient to make it fill up with membrane-bound vesicles? Would you send us to the couch or to a padded cell? Not so fast, as this is precisely what a group of sixteen investigators from three continents have recently shown by expressing caveolin‑1 in E. coli. Caveolin‑1 is the main protein involved in the formation of mammalian membrane structures called caveolae. The consequences were amazing. Caveolae, by the way, are flask-shaped pits in the cell membrane involved in the formation of endocytic vesicles and aspects of cell signaling and lipid metabolism. They are the sites where some bacteria and viruses first attach prior to entering the cell.
Figure 1. An E. coli cell replete with caveolins.(A) Time-course: t0, beginning of protein expression, labeling is associated with the inner membrane (small arrowheads) and occasionally with invaginations (see inset; large arrowhead). Intracytoplasmic vesicles are clearly evident after 30 min to 90 min, until after 120 min the entire cytoplasm, in many cells, is filled with vesicles. Inset in 12 hr expression: anti-MBP immunoblot showing protein levels at individual time points. (B and C) High-pressure fast-frozen, freeze-substituted, and Epon-embedded E. coli showing an h‑caveola undergoing fission from the cytoplasmic membrane (indicated by arrowhead). (D and E) Cells expressing MBP-caveolin‑1 with entire cytoplasm filled (D), and higher magnification of vesicular structures in the cytoplasm (E). (F) Detail of high-pressure fast frozen, freeze substituted E. coli expressing MBP-caveolin1 showing cytoplasmic vesicles. Scale bars: (A and D) 200 nm, (A, inset, E, and F) 100 nm, (B and C) 20 nm. Source
The E. coli used here performed spectacularly, beyond any researchers' dreams. They made bilayer-bound caveolae-like structures that match in key ways the caveolae in animal cells. In fact, they made so many that within a few hours the cells became utterly filled with these vesicles – wall-to-wall caveolae. The manipulations were straightforward. The researchers cloned the caveolin‑1 gene in a standard plasmid expression vector and introduced this construct into E. coli. Cultures of the transformed strain grew vigorously to high overnight cell densities, the same as cells carrying an empty plasmid. By this time, the cells were literally filled with the vesicles. Other than that, little is known as yet about how this affected the bacteria
Next, these researchers asked if it's possible to introduce foreign constituents into the E. coli caveolae. After incubating 'E. coli K‑12 subsp. caveolaefaciens' with fluorescent membrane-staining dyes that can cross the outer but not the inner membrane of Gram-negative cells, they saw lots of fluorescence within the caveolae. So yes, the caveolae contain material from the periplasm. And that material can include proteins. One could call this process endocytosis-like. Something similar has been reported only once in bacteria, to wit in the somewhat enigmatic Planctomycetes (a point that is being debated here).
This filling of bacterial cells with extraneous vesicles is simply startling. We know of no precedent. What this tells us about the plasticity of bacterial cells remains to be seen, but it sounds like there is quite a message here.
Also exciting is that this system may be of use in biotechnology. Consider the possibilities for drug delivery offered by encapsulation of small molecules and proteins introduced into the periplasm. In another proof-of-principle step, these researchers showed that caveolae containing GFP and an immunoglobulin-binding domain from staph Protein A not only became bound to human breast cancer cells carrying an antibody on their surface but appeared to become internalized.
2. Converting a harmless E. coli K‑12 into a potential pathogen
Here is another unexpected event resulting from a mutation in a single gene. E. coli K‑12 is a strain widely considered so safe that one could take a bath in it and even swallow it. Nobody has disputed this even in the old days of the debate about the safety of recombinant DNA technology. Best we know, no instance has been reported of this strain being infective. Therefore, it came to a complete surprise when Koli et al., researchers at the Institute of Molecular Medicine in Delhi found that a mutant in the histone-like DNA-binding protein HU expresses such pathogenesis factors as curli fibers, the lysozyme inhibitor Ivy, and hemolysin E. This happens because a mutation in HU causes major changes in the transcription of many genes, the above included. With these virulence genes awakened, the HU mutant disrupts phagosomal function and is invasive to host cells in culture as well as in a mouse ileal loop model. In addition, once internalized, the bacteria caused a novel form of host subversion, namely the impairment of host cell apoptosis. The effects on transcription are attributed to changes in the supercoiling of the DNA by the mutant protein. The protein induces positive supercoils, which alter the availability of promoters, thus changing the cells transcriptional profile in one fell swoop.
Figure 2. Invasion of intestinal epithelial cells by HU-modified E. coli. Transmission EM of cells infected with mutant (SK842) and wild type (MG1655) at an MOI of 100. Source
Granted, this is not your run-of-the-mill mutation that affects a single function, but it is quite amazing that a broad change in transcription should reveal the potential action of otherwise dormant pathogenicity factors. As the authors said: "Using the E. coli K‑12/HUα(E38K, V42L) variant as a model, we propose that traditional commensal E. coli can adopt an invasive lifestyle through reprogramming its cellular transcription, without gross genetic changes." Keep in mind that, in general, invasion of host cells is not what E. coli strains, including pathogens, usually do.
Let's consider this in context. Many a pathogen can be converted into a harmless organism by a single mutation, so why not the converse? This would happen if you reverted a mutation that inactivates an important toxin. But the situation here is far more complex. The reason the K‑12 strain is innocuous is not due to several of the factors needed for virulence being absent but because they are hidden. A single mutation in an architectural protein (well, in fact a double mutation but still in one gene), one that modifies DNA structure, can have the pleiotropic effect of 'waking up' these virulence genes simultaneously. Perhaps this may not seem far-fetched, but we think that it lies outside the range of what the usual imagination allows.
3. How to Make a Photosynthetic Bacterium in A Simple Step
(modified from a previous post) Try turning an organism into photosynthesizer in one simple step. Seems wildly implausible, no? Photosynthesis as we know it evolved over a long time via a series of complex and sophisticated evolutionary events. Making chlorophyll alone, leaving alone the rest of the photosynthetic machinery, took much doing. Can you imagine changing a "regular" bacterium into a photosynthesizer in a simple direct way? The answer is startling: this miracle can indeed be carried out in one humble stride. Researchers coated a fairly ordinary bacterium with beads of cadmium sulfide and, presto, it now uses light to enhance its ability to fix CO2. You have now provided the bacterium with its own solar panels. The chosen non-photosynthetic bacterium, Moorella thermoacetica, belongs to a class called the acetogens, who can fix CO2 to make acetate, so it's evolved to utilize CO2, albeit without light. This trick may have industrial applications, some even green.
Figure 3. M. thermoacetica–CdS reaction schematics. (A) Schematic of the M. thermoacetica–CdS hybrid system, proceeding from the growth of the cells and bioprecipitation (loading) of the CdS nanoparticles (shown in yellow) through photosynthetic conversion of CO2 (center right) to acetic acid (right). (B) Pathway diagram for the M. thermoacetica–CdS system. Two possible routes to generate reducing equivalents, [H], exist: generation outside the cell (dashed line) or generation by direct electron transport to the cell (solid line). Source
The steps in making the artificial photosynthesizer are simple: Feed the bacteria Cd+2 and cysteine, and they come out of solution as CdS precipitates. The bacteria become beautifully decorated with CdS nanoparticles, about 10 nm or less in size. Now, turn on blue light. When CdS absorbs a photon, it produces an electron and a "hole pair," e– and h+. The electron provides reducing power, which is passed on to the Wood-Ljungdahl pathway to synthesize acetic acid from CO2. The yield of acetate is 90% based on the initial cysteine concentration, the rest becoming biomass. The quantum yield was 2.4% of incident light, comparable to that of plants.
As one of the authors of the paper, Peidong Yang, said: "Our hybrid system combines the best of both worlds: the light-harvesting capabilities of semiconductors with the catalytic power of biology. In this study, we've demonstrated not only that biomaterials can be of sufficient quality to carry out useful photochemistry, but that in some ways they may be even more advantageous in biological applications."
4. All One's Eggs In One Basket
(modified from a previous post). At the heart of every cell's 'operating system' you find the ribosomes, these ingenious nano-machines that translate messenger RNA into all kinds of proteins the cell needs to build and maintain itself. Intuitively, you would probably guess that the genes encoding such vital parts of the 'operating system' are located on the chromosome, the hard drive. They are, in almost all known cases in archaea and bacteria alike.
Figure 4. USB flash drive (aka thumb drive). Customized from: Source
Enter Aureimonas sp. AU20 and its cousins, humble members of the Rhizobiales subfamily of the ubiquitous Alphaproteobacteria. Recently, Hisayuki Mitsui and co-workers isolated Aureimonas from the stem of a soy bean plant and found to their surprise that it lacks rRNA genes on its chromosome. Instead, a typical bacterial rRNA operon is carried on a small (9.4 kb) multicopy plasmid (18 ‒ 34 copies/cell) that is also present in the 4 cousins (out of 12) of Aureimonas that lack chromosomal rRNA genes, too (Figure 4). Thus, it's apparently a family trait, and not a mere genetic accident.
The plasmid does not encode factors known to be needed for plasmid segregation and may rely therefore solely on stochastic distribution of plasmid copies among daughter cells. Furthermore, the authors could not detect any of the known addiction modules that usually ensure plasmid maintenance in the host. Most likely, the selective pressure to stably maintain the rRNA-encoding plasmid is sufficient: you better keep your thumb drive plugged in! But this strikes us a dangerous existence. Lose the plasmid and you are a goner!
You may wonder whether there aren't any viruses lurking here. And you would be right! A virus DNA recently isolated from a marine sponge contains a bacterial 16S rRNA gene. Since marine sponges usually live in consortia with bacterial epibionts and symbionts, it is unfortunate that the authors don't specify whether this 16S rRNA gene was carried by phage or by a eukaryotic virus of the sponge. In any case, this provides another example for an 'operating system on a thumb drive'.
Does this unexpected location of rRNA genes on a plasmid mean that these genes participate in horizontal gene transfer? There is some evidence that this is in fact the case. If so, rRNA-based phylogenies ‒ the marvelous idea that allowed Carl Woese to propose his three-domains model ‒ may turn out to be really messy, in some niches at least. That would then be the normal state of affairs in most of biology.
5. A Bacterium with A Collection of Distinct Nucleoids
(modified from a previous post). We suppose that if anything characterizes this blog it's an emphasis on strange or at least unexpected stories that we encounter along our microbiological peregrinations. But, in our opinion, few matches one related by Christoph under the title A Lakeside Tale. Here he described a very large swamp-dwelling bacterium, Achromatium oxaliferum, that seems to thumb its nose as a basic tenet of biology, namely that polyploid cells contain identical or nearly identical nuclei (or, if prokaryotic, nucleoids). Ha! The cells of A. oxaliferum contain perhaps 200 chromosomes, many of which are different from the others. So, each cell contains a "community genome!" A new biology?
A. oxaliferum is the largest bacterium known from non-marine environments, on average 15 × 50 μm in size (Figure 5). Grossart and coworkers found that the DNA of 27 handcked cells showed considerable diversity (this a amounts to having a hypervariable region). Most surprising is that this heterogeneity was found in the DNA of single cells! The authors checked 131 markers whose genes are in most cases present as single copies in the genome. They found that the majority had 15 – 20 sequence variants. Once again, this is a situation one would readily accept for samples containing members of a taxonomic gemisch, but not for a single 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. Their neighboring genes were also varied. Thus the genes could not be assembled into a single genome.
Figure 5. 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'. Source
The genes of this 'community genome' are phylogenetically related on the genus level, and distributed over a cohort of ~200 chromosomes within single Achromatium cells. This is not a case of polyploidy, a term reserved for multiple copies of the same sequences. If this 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 proportional to its strangeness".
I (Christoph) was recently allowed to peek at Achromatium cells through a microscope, notably at just 6× magnification, I saw a few Euglena flagellates buzzing around, some other tiny unicellular euks, and several dozen 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. The cells were apparently doing well since many were busy with cell division in a very regular manner, that is, constricting at midcell. I could have easily picked single cells – as they did 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 been unable to count their calcite "potatoes". For a microbiologist trained to observe his 'objects of desire' under the microscope at >400× magnification, it was, admittedly, a stunning experience to actually see individual bacteria with the naked eye (ok, equipped with eyeglasses in my case ).
