Small Things Considered

E. coli Cells Face FACS and Get Back into Shape

by Kimberly K. Busiek

Figure 1: The importance of size and shape to the animal kingdom. (A) A well-adapted giraffe enjoys the fruits of its evolutionary labor. (B) Beak variations among Darwin’s finches. (C) A bonobo uses his opposable thumb to dig for termites.

There’s no question that variation in size and shape has conferred selective advantages over the course of evolutionary time. One of the most obvious examples is the long neck and legs of the giraffe, which allow it to snatch foliage that is unreachable by vertically challenged competitors. The variable beak shapes and sizes of Darwin’s finches represent the diverse tool set that evolved when only certain food sources became available. And the appearance of the opposable thumb, a simple change in hand shape, undoubtedly influenced the course of human history.

Pictures Considered #3. How Do You Know There Is a Nucleoid?

by Elio

A time-lapse series of E. coli growing on agar containing 20% gelatin. The time is shown in the lower left corner. The contrast in these photos was reversed to make it easier to see the nucleoids. In actuality, the nucleoids look pale and the cytoplasm dark. Source.

What is more commonplace than saying that prokaryotic cells possess a nucleoid? It is implicit in the term prokaryote itself. Still, it was not shown definitively until the 1940s that bacteria and archaea have such differentiated structures made up of condensed DNA. It was the careful work of “bacterial cytologists” (as they were then called) and especially that of the eminent Carl Robinow that furnished convincing evidence for the nucleoid’s existence. Bacterial cells, then and now, were commonly prepared for microscopic examination by fixing with heat and staining with basic dyes. Such dyes stain more intense than acid ones because bacteria are replete with acidic groups from the RNA in the ribosomes. So abundant are these RNAs that these dyes stain the cells uniformly, and the nucleoids are not apparent. To reveal the nucleoids, cells were treated with ribonuclease or with dilute acids that selectively degrade the RNA and then stained. The DNA-containing nucleoids retain the dye and are now easily seen. When thin sections of bacterial cells could be examined under the electron microscope, analogous structures were seen, thus providing additional evidence that bacterial nucleoids exist. And yet, a small lingering doubt remained.Both of these methods looked at chemically treated (“fixed”), dead bacteria. Could this lethal treatment create artifacts regarding the observed “nucleoids”?

Holey Biofilm!

by Gemma Reguera

Source.

As a child, I was always fascinated by the holes (or eyes) in Swiss cheese, always inspecting the tunneling system before getting a good bite. Although the holes are the result of microbial activity (the accumulation of CO2 released by fermentative bacteria), I bring up the Swiss cheese analogy for very different reasons. Try to picture a similar landscape of tunnels and holes in a bacterial biofilm. And that’s what today’s story is about … a ‘holey’ biofilm.

Poking Holes in Biofilms

B. thuringiensis biofilms are pierced with holes and tunnels created by the planktonic swimmers. The arrow points at a large hole formed by the collective motion of a chain of cells. (Scale bar, 20 μm). The trajectories of the swimmers that lead to the formation of the biofilm holes is shown on the right panel. Source.

In a recent study published in PNAS, Houry and collaborators used time-lapse microscopy to monitor the biofilms formed by the bacterium Bacillus thuringiensis and noted that a small subset (0.1 to 1%) of all the cells in the biofilm were motile. The rest of the cells were sessile and immobile except for some minor oscillatory motions hampered by the surrounding biofilm matrix. The swimmers infiltrated the biofilms in all directions, creating a landscape of tunnels and holes like in Swiss cheese. By tagging planktonic cells (that is, cells growing free in the surrounding liquid) with the green fluorescent protein (GFP), the authors showed that the biofilm swimmers were in fact planktonic cells. The swimmers infiltrated the biofilms independently of the flow dynamics of the surrounding fluid and their tunneling activity was exclusively dependent on the rotational activity of their flagella. Despite the biofilm barrier, the swimmers had average velocities as high as 7.3 μm/s in young (24 h old) biofilms. For a movie showing these rapid motions, click here. The swimming velocities decreased progressively as the biofilms aged, with the lowest velocities (4.2 μm/s) being measured in the oldest (72h old) biofilms. This is because the biofilm matrix also becomes more dense and rigid over time (and, therefore, more difficult to permeate). Still, these speeds are remarkable for cells that are swimming through a biofilm matrix!

Pictures Considered #2. The E. coli Chromosome Caught in the Act of Replicating

By Elio

A radioautograph of the E. coli chromosome in the act of bidirectional replication. The image shows a circular molecule with two “rabbit ears”, representing the newly replicated regions. E. coli DNA was labeled with tritium-labeled thymidine by growing the cells for two generations in the presence of this specific DNA precursor. Cairns had developed a method for depositing intact DNA molecules on a microscope slide, then covering it with a photographic emulsion. The slide was then stored in dark for two months to allow the tritium to decay and sensitize enough photographic grains to reveal patterns after photographic development, as seen in this radioautograph. The insert on the right corner of the image shows a tracing of the replicating chromosome, which is a circle nearly 1 mm long. From Cairns, J.P.: Cold Spring Harbor Symposia on Quantitative Biology 28:44, 1963.

We continue our series of images that have made a difference in microbiology. This one, published by John Cairns in 1963 has earned a most deserved place in textbooks and reviews. It shows a radioautograph of an intact E. coli chromosome in the act of replicating. From this image, Cairns concluded that the E. coli chromosome is:

• a single piece of double stranded DNA, 700-900 μm long, and
• circular.

These truths had been surmised from other, mainly indirect pieces of evidence. For instance, the idea that the genome consists of a single chromosome had already been inferred from the fact that only a single genetic linkage group could be found in E. coli. Its circular nature had been suspected because all the genes on the chromosome were circularly permuted. But this image gave these notions physical authenticity, ergo finality. Such important conclusions from one single picture!

In addition, by labeling the newly replicated DNA in the chromosome with a short pulse of tritiated thymidine, Cairns provided conclusive evidence for the bidirectionality of chromosome replication, with each of the two replicating forks containing one new and one old DNA strand.

Correction

As pointed out by Christoph Weigel and David Lane, I wrongly attributed to Cairns the evidence that the E. coli chromosome replicates bidirectionally. In fact, the definitive proof stems from a later elegant radioautographic study by Prescott and Kuempel and several equally convincing genetic experiments, e.g., by Masters and Broda. A regrettable memory lapse…..

Two grain tracks produced by E. coli chromosomes from cells labeled for 13 min with [³H]thymine, followed by labeling for 2.5 minutes with [³H]thymine and [³H]thymidine.  The numbers indicate the length of the grain tracks. Each track is produced by two daughter (sister) DNA duplexes. Source.

A PomZ Scheme For Turning One Cell Into Two

By William Margolin

A pyramid scheme applied to academia. Source.

The division of one cell into two daughter cells is the crux of biological reproduction. But how do cells determine where along their dimensions division will occur? For bacteria, the best-studied species for basic biology, including cytokinesis, are the old standbys Escherichia coli, Bacillus subtilis, and Caulobacter crescentus, mainly because of their easy cultivation and genetic tractability. 

But, given bacterial diversity, the lifestyles of these representative workhorses aren’t necessarily the norm; they can even be relatively boring compared with the habits of others like the myxobacteria. One such species with exceptional behaviors is Myxococcus xanthus, whose cells travel in packs, eat other bacteria, and differentiate into fruiting bodies when their food runs out, eventually forming spores. Of course, in addition to moving around and eating a lot, myxobacterial cells still need to divide. Recent results from Lotte Søgaard-Andersen’s group and their collaborators show that the way myxobacterial cells select where to split into two is also out of the ordinary. 

Bdellovibrio’s Appetite for Metabolites

by Henry N. Williams

The Poly-hydroxyalkanoate repeating unit of PHA. (no source)

Not long ago, Elio said in this blog that predation, a major force in evolution, is somewhat neglected in microbiological circles. The full implications of predation are just beginning to be uncovered as more becomes known about the ecology, physiology, and genomics of predators against microbes and their interactions with their prey. The collective knowledge of how they influence each other will naturally feed curiosity of scientists regarding how some cellular characteristics evolved. Here, I address a new metabolic feature of a predatory bacterium that will certainly lend itself to evolutionary considerations.

The Slime That Smiles

by Heidi Arjes

Figure 1: Cell death occurs between the emerging digits during development. A fluorescent chemical was used to visualize regions of cell death in these embryonic mouse limbs. Source.

Have you ever wondered how individual fingers form? If you have taken a developmental biology class, you know that the hand first develops as a mitten-like structure with the future fingers connected (Figure 1). Later, during normal development, the cells in the areas between the fingers undergo programmed cell death, and thus leave behind five fingers on each hand. Programmed cell death is important not only in development and long term viability of animals, but also plays a role in unicellular organisms. For example, in the social amoeba Dictyostelium, a subset of cells undergoes a type of programmed cell death similar to autophagy to form fruiting body stalks. The remaining dead cells in the stalk are highly vacuolated and provide support for the spore body (figure 2).

A Bacterium Learns Long Division

by Nanne Nanninga

The common picture of a dividing rod-shaped bacterium encompasses the positioning of the divisome, including an FtsZ-ring, in the cell center. This occurs after the cell has doubled its length without increasing its diameter. Conversely, increase in diameter without cell elongation would seem highly unlikely in a rod-shaped organism. Yet, this happens.

Figure 1: SEM of tightly apposed ectosymbionts on the surface of L. oneistus. Electron micrograph by N. Leisch.

In fact, this is the normal condition for an ectosymbiotic bacterium that lines the surface of the marine nematode Laxus oneistus. The symbiont is a g-proteobacterium like E. coli, but unlike E. coli it has not been cultured outside its natural habitat. As originally described by Polz et al. in the early nineties, the bacteria are positioned perpendicular to the surface of L. oneistus. They are glued to the surface of the nematode by a C-type lectin. The original paper already indicated that division takes place longitudinally, with one of the daughters presumed to remain attached to the nematode. This makes sense because otherwise the daughter cells would get lost to the environment. Recall that this phenomenon lies at the basis of the Helmstetter-Cooper baby machine, whereby one of a pair of newborn cells is selected to start a synchronous culture (Helmstetter et al.)

Recently, Leisch and colleagues have extended these observations by carefully determining cellular dimensions and visualizing the FtsZ division protein with fluorescent E. coli monoclonal antibodies. The results can be compared with E. coli data (Figure 2A, B). Whereas E. coli elongates as expected, this is not what happens with the symbiont. The symbiont does not change its length (Figure 2A) but increases its diameter (Figure 2B). Consequently, the symbiont divides longitudinally, (Fig. 2 C-H). Immunostaining of FtsZ reveals that FtsZ positioning correlates with the cell constriction. In fact, an ellipsoidal FtsZ-ring is observed stretched along the length of the endosymbiont.

Cell Division Through DNA Curtains

by Gemma Reguera

Despite the apparent simplicity of bacterial cells, their cell division cycle is a complex developmental program that couples cellular growth to the replication and segregation of chromosomes and the division of the cell’s cytoplasm (aka cytokinesis) (Fig. 1). The bacterial cell division cycle starts with the commitment of the cell to reproduce. This is the step in which, forgive the pun, size truly matters. During active growth, the cell’s size changes to accommodate increases in mass, volume, and biosynthetic capacity. When the cell reaches a critical size, essential cellular functions such as intracellular transport and nutrient uptake are constrained and optimal growth can no longer be supported. Provided conditions are adequate for growth, cell division is undertaken to allow the bacterium to regain its individual cellular fitness.

Fig. 1: As the cell grows in size, the chromosome is replicated and segregated. A constricting septum forms at midcell to divide the cell’s cytoplasm and produce two daughter cells. Source.

Living On the Edge…of the swarm

by Gemma Reguera

The edge of a wave, a tsunami wave that is. Source.

If anybody knows how to move, it’s bacteria. They swim in liquids using rotating flagella, but they also know how to twitch, glide, and slide on surfaces. The mechanisms that power their surface motility are varied, ranging from energy-intensive processes such as the extension and retraction of type IV pili (twitching) to movement via focal-adhesion complexes of the cell’s outer surface (gliding) or growth-induced translocation aided by surfactants (sliding). Some flagellated bacteria can also use the rotating motion of the flagellum to move across moist surfaces, a process known as swarming motility. As the word indicates, swarming is a collective behavior. Swarming cells move side-by-side in regular or irregular formations known as rafts or swarms. See, for example, previous blog posts showing the regular rafts produced by Paenibacillus or the distinctive, irregular terraces formed by Proteus. And even E. coli does it, too!

Despite their close proximity, the cells within the swarms do not attach to each other, nor do they adsorb to the surface. They swim across a thin film of liquid that is drawn out of the underlying surface; they rely on random collisions with neighboring cells to reorient themselves and to coordinate the collective motion (pretty much like the random movements of sheep within a flock). Cells at the swarm’s outermost edge truly live…well…on the edge, stuck between the liquid boundary and the force of the spreading swarm. Time-lapse microscopy shows that most of these cells slow down and even stall. Sometimes they reverse their direction to face inwards and become stuck to the substratum. However, these cells eventually get free and swim back to the center or along the edge of the swarm, suggesting that they continue to rotate their flagella while stalled. But could this be proved?