Moselio Schaechter


  • The purpose of this blog is to share my appreciation for the width and depth of the microbial activities on this planet. I will emphasize the unusual and the unexpected phenomena for which I have a special fascination... (more)

    For the memoirs of my first 21 years of life, click here.

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April 14, 2014

By Chance and Necessity: The Role of the Cytoskeleton in the Genesis of Eukaryotes

by Daniel P. Haeusser

How did Eukaryotes Evolve from Prokaryotes?

Figure1 Figure 1. The mysterious root of the eukaryotic origin. Evolution of prokaryotes into eukaryotes undoubtedly involved mitochondrial acquisition, but the full details of the story are far from certain. Source.

One of the most exciting and enduring obscurities of biology lies in the early stages of the evolution of “our” eukaryotic cells (Figure 1). The endosymbiotic theory accounts well for the present existence of the mitochondrial and chloroplast organelles of eukaryotes. Although there is evidence for present day inter-bacterial endosymbiosis (also see here and here), the details of the route leading to the establishment of organelles remain enigmatic.

Even murkier is the question regarding the origin of the nucleus (Figure 2). While prokaryotic cells with organelles arguably exist and scientists have identified Planctomycetes that enclose their DNA with internal membrane continuous with the cell membrane, a truly independent membrane-bound nucleus prevails as the defining hallmark of the eukaryotes. (A recent report even calls this textbook difference into question! However, another study calls these conclusions into question.)

Unlike the clear bacterial origin of the mitochondria and chloroplast, it turns out that no single existing model has received broad acceptance to explain the existence of the nucleus. Scientists have speculated that it could have arisen from an endosymbiotic event between an archaeon and a bacterium (though who engulfed whom is also uncertain). Some have proposed that an autonomous internal gathering of cell membrane as in Planctomycetes formed the eventual independent nuclear organelle, and yet others have even suggested infection by an enveloped virus as playing a role.

Figure2 Figure 2. Two of the prevailing models for the origin of the nucleus. Source.

Not only are details on the evolution of these eukaryotic innovations (nucleus and mitochondria or chloroplast) shrouded in mystery, but just as unclear is the temporal relation between their developments. Which came first, the nucleus or the mitochondrion?

A recent online article by Ed Yong for Nautilus Magazine gives a comprehensive summary of one of the prevailing hypotheses proposed to answer that question. The general crux is that the relatively sudden marriage of two prokaryotes into one stable individual cell occurred just once in the evolutionary history of life on Earth, and that allowed the emergence of an entire new branch on the tree of life. Here, the engulfment of a bacterium by an archaeon permitted the development of mitochondria. This union augmented and expanded the metabolic and energy generation capabilities of the new organism, thereby permitting genome size expansion and organelle proliferation (compartmentalization). All this gave sudden rise to the establishment of the hugely differentiated eukaryotic lineage.

Figure3 Figure 3. Labeled eukaryotic skeleton (Bovine pulmonary artery endothelial cells). Microtubules are shown in green and actin in red. DNA in the nucleus is shown in blue.

Some pieces of evidence detailed in the Nautilus article are consistent with a model where mitochondrial acquisition ‘licensed’ the genesis of eukaryotes. Yet, the jarring thing about this mitochondria-centric hypothesis is that it says nothing about the nucleus. The big problem with mitochondrial acquisition alone as the driving force of eukaryogenesis is that such a development is not a simple matter of metabolic and energy concerns. As recognized in Yong’s article, prokaryotes also largely lack eukaryotic ‘architecture’, leaving them “forever constrained in size and complexity,” an idea also discussed in an essay by Kevin Young. The mitochondrion alone is not a ready system for getting things around a cell as it increases in size and complexity. The cell would still lack the architecture and machinery to be released from the limits of simple diffusion, a significant barrier between the small prokaryotes and the (usually) larger eukaryotic cells.

In short, the mitochondria-centric hypothesis for eukaryotic genesis ignores the important uniqueness of the eukaryotic cytoskeleton (Figure 3). So, let’s take the cytoskeleton into consideration.

Why Are Prokaryotes Different from Eukaryotes?

A superb, recent essay by Julie Theriot addresses these issues with a focus on comparing eukaryotes to bacteria (not to archaea). Theriot’s essay probes deeply and meaningfully into the question of ‘why are prokaryotes and eukaryotes different’. With expertise in the cytoskeleton, her ruminations on this ‘why’ question rest on that dynamic architecture of eukaryotes that permits them to reach proportions of growth and organization rarely seen in prokaryotes.

To summarize her major points: While we now know that the cytoskeleton is present and broadly conserved across all domains of life (with diverse proteins in the actin, tubulin, and intermediate filament families), bacteria notably lack a special class of cytoskeleton-related proteins: the nucleated filament assembly factors and the motor proteins. Eukaryotes make ample use of these tools. By providing a mechanism for polarized assembly that permits motor protein directionality and inherently oriented filament growth, the eukaryotic cytoskeleton is the architectural machinery that achieves functions such as chromosome movement, vesicle and organelle transport, and endocytosis. These, in turn, help the cell attain sizes and complexities that otherwise would be unattainable.

Figure4 Figure 4. Some of the techniques evolved by pathogenic bacteria to subvert the host cell cytoskeleton using bacteria-derived secreted eukaryotic cytoskeletal regulators. Source.

Theriot correctly points out that, in theory, bacteria are capable of making such cytoskeleton-related factors. Indeed, several pathogens contain genes for proteins that alter and control cytoskeleton assembly (e.g. here), including through nucleation (e.g. here). But bacteria export these proteins for use on the host(Figure 4), never (that we know of) for their own cytoskeleton. So why didn’t they learn to use their cytoskeletons in the same way to gain some of the benefits that the eukaryotes enjoy? Why not follow the eukaryotic route?

Possible answers to these questions receive due consideration in Theriot’s essay, but what I found most fascinating is her observation that bacteria have a trove of genes that encode different varieties of the major cytoskeleton protein families, particularly with actin homologs. It seems as though bacteria have evolved one distinct homolog for each cellular function. Eukaryotes, by contrast, mostly contain just one type of each family member, which is then specifically regulated to perform several distinct functions. Could this explain why bacteria don’t use their cytoskeleton like eukaryotes?

With Theriot’s essay and the Nautilus article in mind, I wondered whether the existing unique properties of the eukaryotic cytoskeleton could fit into models for those cryptic early steps in the evolution of eukaryotes from prokaryotes. So, let’s circle back to the question of eukaryogenesis, but now with the cytoskeleton in mind.

“Every Act of Creation is First an Act of Destruction” – Pablo Picasso

What if the first endosymbiotic merging that permitted the outburst of the eukaryotic lineage were not that of mitochondrial acquisition (a bacterium engulfed by an archaeon), but rather the reverse, something more akin to what has been considered a possible origin of the nucleus, the engulfment of an archaeon by a bacterium.

Theriot’s essay avoids discussion of archaea, perhaps because we know precious little about them. In visual appearance, archaea and bacteria are look-alikes, but archaea contain genetic and mechanistic facets that are both bacteria-like and eukaryote-like. The cytoskeletons of archaea are similarly mixed: while many encode bacterial components such as ftsZ, their overall cytoskeleton composition is closer to the eukaryotes’. Crenactin, for instance, is phylogenetically nearest to canonical actin rather than to the myriad actin homologs found in bacteria. We don’t know enough about archaeal cytoskeleton control yet to figure out if they have nucleated assembly factors, motor proteins, or any of the other unique properties of the eukaryotic cytoskeleton. But given the presence of other rudimentary eukaryotic systems in archaea, such as ubiquitination, it's reasonable to hypothesize that some archaea may employ and control their cytoskeletal architecture in ways closer to those of eukaryotes.

So then, imagine if a bacterium with its cytoskeleton composed of multiple, monofunctional actin/tubulins were to engulf an archaeon with a relatively simpler cytoskeleton consisting of a single member of the actin and tubulin families each. Were the engulfed archaeon capable of growth and further replication within its new host, some cells would die with time, leaking archaeal contents into the bacterial host cytoplasm. I propose that this could include ‘eukaryotic-like’ cytoskeletal proteins and regulatory system components like proto-motor proteins and nucleation factors.

The presence in the same bacterium of multiple actin and tubulin homologs carries the risk of cross talk between the regulator and those homologs. If so, this may preclude or at least discourage the evolution of task-specific regulators that affect assembly like nucleation proteins, or factors like motor proteins. Thus, if the cell needs to nucleate one actin homolog involved in plasmid segregation, it may not want to have to nucleate the other actin homolog, e.g. one involved in maintaining cell shape. Unique factors would then need to evolve for each cytoskeleton homolog, a demanding task. On the other hand, the ‘single member’ system of the archaea may allow for the innovation of these kinds of regulatory systems. They could comprise a small set of regulators whose activity can be modulated depending on where and when they’re needed. Inside their bacterial host, these archaeal regulatory factors are now free to act via crosstalk on the bacterial homologs.

This potential for disastrous cross-talk of a single regulator on different homologous cytoskeleton family members in a single cell would also explain why several bacterial species appear perfectly capable of evolving eukaryotic-like cytoskeleton control for export during pathogenesis (in trans), yet eschew adoption of such systems in cis. Their relatively broad repertoire of highly similar cytoskeletal components simply makes it too risky a proposition.

Figure5 Figure 5. (Left) Pablo Picasso’s Violon (Violin) 1911-12, Kröller-Müller Museum, Netherlands. Just as the audience’s image of a violin is destroyed through deconstruction within the oval frame to achieve Picasso’s art, so too may a bacterial cytoskeleton have been disrupted under the influence of archaeal-evolved factors following endosymbiosis. (Right) Chance and Necessity by Jacques Monod. Borrowing Monod’s title, the chance merging of two distinct systems for cytoskeletal regulation may, by necessity, have influenced eukaryogenesis.

The Picasso quote that titles this section could be equally apt for this proposed version of eukaryogenesis (Figure 5, left). To summarize and repeat: The chance mixing of two distinct modes of cytoskeleton regulation in merged organisms (one with many similar members with unique functions and the other with single members with broad functions) would potentially result in a war for control. Because of regulatory crosstalk, one system would need to eventually win out. Perhaps the products of the endobacterial archaeal genome interfered with, and ultimately destroyed, the bacterial cytoskeletal system, creating a novel one in its place.

An archaeal cytoskeleton with rudiments of nucleation and motor proteins, but now within a bacterium with its own unique genes for metabolism, creates a novel organism with the architectural system needed for further evolution. This could include the compartmentalization of a newly merged genome under the control of an archaeal-derived cytoskeleton. With the acquisition of energy-generating organelles and further compartmentalization, larger physical dimensions and a bigger genome size could be attained. More stable, these early eukaryotes would displace any amitochondrial species and, with a finer tuning of their cytoskeleton, develop more innovations.

According to this model I’m proposing, the key event in the eukaryogenesis is, to reference Monod (Figure 5, right), the chance merging of two systems by the necessity of regulatory cross-talk gaining dominant control over homologs of the other system. Above all, such a model predicts that we should be able to find existing archaea with genes that encode nucleation factors and/or motor proteins used to control the functions of its own cytoskeleton. To my knowledge they have not yet been found, but I don’t think that we’ve looked particularly hard either.

Are there problems with this line of thinking? Are there other considerations? Most importantly, what kind of experiments do you think would currently be possible to address these speculative models? The mystery of eukaryogenesis still has a lot of questions, and like with many things in science, I don’t think that our curiosity will soon be satisfied.

Daniel




Daniel is a postdoctoral fellow in the Margolin Lab in the Department of Microbiology & Molecular Genetics at the University of Texas, Houston Medical School. He also teaches as an adjunct professor at the University of Houston-Downtown in the Department of Natural Sciences.

March 17, 2014

The Bacterial Chromosome: A Physical Biologist's Apology. A Perspective.

by Suckjoon Jun

I entered the bacterial chromosome field in 2004 as a fresh Ph.D. trained in theoretical physics. Ten years is not long enough for one to gain the depth and breadth of a scientific discipline of long history, certainly not for an early career scientist to write an essay of the status of A Mathematician’s Apology (Hardy 1940). Nevertheless, I agreed to write this Perspective as a physicist who entered biology, because my colleagues are often curious to know what drives physicists to become (physical) biologists, and make them stay in biology despite many challenges. I also wanted to share several lessons I have learned because, while some of them are personal and specific to my field, I have a good reason to believe that they might resonate with many future travelers. This Perspective is for them.

I would like to start with the story of one of the most familiar and yet mysterious forces in nature—gravity. Galileo is said to have dropped two balls of different masses from leaning Tower of Pisa in Italy some five hundred years ago. His experiment was to demonstrate that, on the contrary to Aristotle’s theory, the falling rate of the balls was independent of their mass. A modern version of this experiment was performed on the Moon by the Commander of Apollo 15 with a hammer and a feather. For a movie of this experience, click here. When released from the same height at the same time, the two falling bodies hit the surface of the Moon simultaneously! On the Earth, however, the feather would have fluttered, as if alive, because of the air.

Initially attracted to the beauty of Amsterdam, I started my post-doctoral research at AMOLF, an interdisciplinary research institute known for exciting interactions at the interface between physical and biological sciences. The forces I was interested in were much less tangible than gravity. In particular, I was supposed to explain the driving force underlying segregation of a replicating chromosome in Escherichia coli. It sounded simple to me, except that I barely knew anything about bacteria, certainly without realizing that it was one of the long-standing problems in biology. I knew the DNA biophysics literature fairly well, but when I saw the beautiful 1992 illustration of E. coli in Goodsell, it was obvious that something like the wormlike chain model was not going to be very useful to understand segregation of the whole chromosome. What worried me was the directionality—if I were a small protein sitting on a replicating chromosome, could I tell which DNA segment belongs to which sister DNA? Physicists like questions like that, whether they are rooted in physics or biology.

Continue reading "The Bacterial Chromosome: A Physical Biologist's Apology. A Perspective." »

February 03, 2014

Adhering To The 'Replicon Model' The Sloppy Way

by Christoph Weigel

Figure1
Figure 1. 'Circles in the Sand'. Source.

Sixty years ago Jacob, Brenner and Cuzin devised their 'Replicon Model', inspiring and useful guideline for replication research ever since. According to the model, a 'Replicon' is a genetic element replicated from a single 'Replicator'—replication origin, in modern terms—and replication is triggered by a positive trans-acting factor, the 'Initiator' (see the sketch). One hallmark of the 'Replicon Model' was the postulation of a positive regulator: at the time of its publication gene regulation was mostly thought about in terms of negative regulation or repression, inspired by the seminal Lac operon paradigm.

A Matter of Language

Many bacteria have a single chromosomal replication origin, oriC, which has been identified and studied in E. coli (Gammaproteobacteria), Bacillus subtilis (Firmicutes), Caulobactder crescentus (Alphaproteobacteria), Helicobacter pylori (Epsilonproteobateria), Mycobacterium tuberculosis and Streptomyces coelicolor (Actinomycetes), to name just some favored model organisms. The 'Initiator' in bacteria is the DnaA protein. All sequenced bacterial genomes have dnaA genes and all DnaA proteins are homologs that belong to a distinct subclass of the AAA+ ATPases. All bacteria employ a set of conserved replication factors for initiation, strand separation, priming, clamping, and discontinuous DNA synthesis. Despite this relative simplicity, the pre- and post-initiation mechanisms that ensure the 'once and only once' chromosome replication per cell cycle turned out to be not only intricate but astonishingly variable among the cases studied. Using a metaphor one might say that with respect to replication, all bacteria speak English, using the same grammar and syntax but each branch with a rather unique local dialect in their vocabulary. Just like a guy from Inverness, Florida would face problems getting along in Inverness, Scotland.

Continue reading "Adhering To The 'Replicon Model' The Sloppy Way" »

October 28, 2013

Programmed Cell Death

by S. Marvin Friedman

Gone are the days when bacteria were thought to just grow and divide and not bother to converse with one another. That simple idea has produced mountains of data and most of what we know about bacterial physiology is based on this notion. It turns out, as we know now, that this is an oversimplification. Lots more goes on in the life of bacteria, much of it dealing with communication with other cells. One widespread, if menacing-sounding, mode of communication consists of killing individuals of a population, apparently for the benefit of other of its members. A prevalent mechanism that accomplishes this is the toxin-antitoxin system for programmed cell death. Here, some seemingly altruistic cells are killed from within whereas others get dispatched after receiving extracellular signals. A lot is known about both mechanisms, this being a very active field of research. The signals involved are the subject of this piece.

Figure1
Figure 1. Toxin–antitoxin (TA) module. Antitoxins bind their cognate toxins and inhibit toxin activity. In E. coli, the toxin is activated by degradation of antitoxin either by the Lon or the ClpAP protease. Activated toxins inhibit cell growth and/or lead to cell death. Source.

The Loaded Gun

Many, possibly most bacterial genomes carry toxin-antitoxin modules consisting of two genes in an operon, one that codes for a stable toxin, the other for a labile antitoxin. When the antitoxin vanishes for whatever reason, the toxin can go to work and kill the cell that made it or, through extracellular signals, induce the killing of adjacent cells. Most bacteria contain multiple versions of these modules. Bioinformatic analysis of the tubercle bacillus genome has come up with an astounding 88 putative toxin-antitoxin candidates!

A widely studied system (and the earliest discovered in a bacterial chromosome) is mazEF in Escherichia coli. Stressful conditions that turn off the expression of the mazEF module result in reduction of antitoxin MazE within the cell, allowing toxin MazF to act freely. Such conditions include inhibition of transcription and/or translation, severe amino acid starvation leading to overproduction of the alarmone ppGpp and DNA damage. Engelberg-Kulka’s laboratory, where this phenomenon was first discovered in the bacterial chromosome (it had been found earlier in plasmids by Yarmolinsky) had shown that mazEF-mediated programmed cell death is triggered in other cells of the population by an extracellular quorum sensing factor, EcEDF (for E. coli Extra-Cellular Death Factor). It was identified as the fairly simple pentapeptide NNWNN and acts as a quorum-sensing molecule. In the present report, Kumar and coworkers now extended this story to other species, investigating the EDFs of Bacillus subtilis and Pseudomonas aeruginosa.

Cross Talk

Prior work had shown that in E. coli mazEF-mediated cell death takes place in dense cultures and that the supernatant from such a culture induced cell death when added to dilute cultures of E. coli. Now, supernatants from B. subtilis and P. aeruginosa were added separately to dilute E. coli cultures that had been treated with rifampin to induce mazEF. Both the B subtilis and P. aeruginosa supernatants killed these E. coli cells. Thus, both B. subtilis and P. aeruginosa supernatants contain EDFs. To purify them, the authors ran supernatants through high performance liquid chromatography (HPLC). The only compound (BsEDF) isolated from B. subtilis turned out to be a hexapeptide with the amino acid sequence RGQQNE. Three active compounds were isolated from P. aeruginosa, the nonapeptide INFQTVVTK (PsEDF-1) and the hexadecapeptides VEVSDDGSGGNTSLSQ (PsEDF-2) and PKLSDGAAAGYVTKA (PsEDF-3). Synthetic identical peptides also displayed EDF activity.

Figure2
Figure 2. The effect of the supernatants from dense cultures of B. subtilis or P.aeruginosa on mazEF-mediated cell death in E. coli. Wild-type and ΔmazEF E. coli cultures were not diluted (Dense) or diluted to a density of 3 x 104 cells/ml in M9 medium (M9) or in a supernatant of a dense culture (SN) of B. subtilis or P. aeruginosa. The samples were incubated without shaking at 37°C for 10 min and for another 10 min with rifampin (10 µg/ml). Source.

Programmed cell death in B. subtilis is mediated by the ydcDE operon that codes for a toxin-antitoxin module and belongs to the mazEF family. The authors found considerable cross-talk, as each of the EDFs from E. coli, B. subtilis or P .aeruginosa added separately to dilute cultures of B. subtilis resulted in ydcDE-mediated cell death. Next, they determined which amino acids of BsEDF are essential to cause cell death. The first (arginine) was not required to kill E. coli, but the second one (glycine) was essential. To kill B subtilis, however, glycine, the third (glutamine), fourth (glutamine) and sixth (glutamic acid) amino acids were absolutely required, and the fifth (asparagine) partially required.

How Does the Toxin Work?

It has been known that MazF works as an endoribonuclease by cleaving RNA and that EcEDF enhances this activity. Would BsEDP and each of the PsEDFs also enhance MazF endonucleolytic activity in E. coli? The authors assayed this activity using a continuous flurorometric assay with substrates consisting of one or five RNA bases flanked by DNA nucleotides labeled with a fluorophore at their 5’ ends and a quencher molecule at their 3’ ends. Processing of such a substrate leads to a great increase in fluorescence. Adding BsEDF or any of the PsEDFs led to a concentration-dependent enhancement of MazF activity. The PsEDFs were, however, less efficient than EcEDF or BsEDF. Which amino acids in BsEDP are critical to stimulate MazF activity in vitro? These workers found that the second (glycine) and fifth (asparagine) amino acids are absolutely required, whereas the fourth (glutamine) and sixth (glutamic acid) are partially required and the first (arginine) and third (glutamine) acids are not necessary. Thus, for both the in vivo effect of BsEDF on cell death in E. coli and the in vitro effects of BsEDF on MazF activity, the second and fifth amino acids (though different) are absolutely required and the first amino acid is dispensable.

Figure3
Figure 3. BsEDF and the PaEDFs enhance the in vitro MazF endoribonucleolytic activity. (A) How the endoribonucleolytic activity of MazF was measured. Cleavage of the chimeric fluorescent oligonucleotides by MazF increases fluorescence emission of the fluorophore FAM. (B) The time course of the effect of BsEDF (RGQQNE) on MazF activity (Left panel). The relative (%) increase of MazF activity induced by BsEDF (Right panel). (C) Ditto for the effect of PaEDF-1 on MazF activity. Source.

Using affinity columns, the authors showed that BsEDF binds directly to MazF but not to MazE. The BsEDF mutant with alanine substituted for glycine in the second position did not bind to MazE. Previously, work from this laboratory showed that EcEDF can overcome the antagonistic effect of the antitoxin MazE on the E. coli MazF toxin, thus freeing the toxin from its inhibitor and enhancing its activity. The way this works, it is thought, is by EDF displacing MazE from the MazE-MazF complex. Would BsEDF and PsEDFs also do this? BsEDF did overcome the inhibitory effect of MazE on MazF, but none of the PsEDFs displayed this activity. However, EcEDF was more efficient than BsEDF in overcoming the inhibitory effect of MazE. The same amino acids of BsEDF were required to overcome the inhibitory effect of MazE and for stimulating the E. coli MazF activity in vitro.

A variant form of EcEDF with the sequence NNGNN (named EciEDF) interferes with the ability of WT EcEDF to stimulate the activity of E. coli MazF. Likewise, the variant BsEDF, RAQQNE (named BsiEDF) interferes with the enhancement of E. coli MazF activity by WT BsEDF. Importantly, they found that iEDFs from each species prevented the activity of the EDF of its own species but not that of other species. This result strongly suggests that EDFs from E. coli, B. subtilis and P. aeruginosa interact at different sites on E. coli MazF.

All In The Family?

In E. coli, the quorum-sensing peptide EcEDF triggers a MazF-induced downstream pathway that leads to death of most of the population, leaving a small subpopulation to survive. It is interesting to note that in contrast to the situation in E. coli and B. subtilis, cell death mediated by P. aeruginosa is population-independent, suggesting that its EDFs are used to kill other bacteria but not itself. The present study of EDFs from B. subtilis and P. aeruginosa indicates that programmed cell death is a much broader phenomenon that also involves interspecies killing and that could play a major role in the regulation of mixed bacterial populations. As the authors say: ” ….the production and the role of the QS EDF peptides in interspecies bacterial cell death predicts the existence of an ‘EDF family’ that would also be found in other bacterial species.” An exciting ramification of this research is the possibility of using EDFs as a novel class of antibacterial agents. Could this spell welcomed troubles for those hard-to-treat multiple antibiotic-resistant strains?

Friedman, Marvin_sm

S. Marvin Friedman is Professor Emeritus, Department of Biological Sciences, Hunter College of CUNY, New York City and an Associate Blogger for Small Things Considered.

ResearchBlogging.org


Kumar S, Kolodkin-Gal I, & Engelberg-Kulka H (2013). Novel quorum-sensing peptides mediating interspecies bacterial cell death. mBio, 4 (3) PMID: 23736285

October 21, 2013

A Bacterial Body Clock: Cryptic Periodic Reversals In Paenibacillus dendritiformis

by Monika Buczek

Figure1

As humans we live our lives in 24-hour increments—waking, eating, and sleeping at specific times dictated to us not solely by our discerning willpower, but also by the greater underlying persuasion of our circadian rhythm. Based on the earth’s rotation from day into night, we have internalized a deeply rooted clock that drives how we behave in response to our genetic expression patterns. It’s not hard to imagine that several other organisms respond to an internal sense of time. Sure enough, the 24-hour circadian rhythm is a highly conserved behavior—from complex mammals down to plants, fungi, and cyanobacteria. Interestingly, there are also examples of different temporal rhythm patterns—ranging from years and seasons to minutes and seconds. A curious example is that of the bacterium Paenibacillus dendritiformis, which seems to have its own internal clock of a mere 20 seconds.

Continue reading "A Bacterial Body Clock: Cryptic Periodic Reversals In Paenibacillus dendritiformis" »

October 14, 2013

Vesiculation: Another Bacterial Skill

by Elio

Microbiology, we will agree, is a vast subject where many important aspects are likely to evade one’s sight. Here’s an example—the formation of vesicles from the outer membrane of Gram-negative bacteria. This phenomenon, known as vesiculation, is widespread and noteworthy for enhancing our understanding of bacterial capabilities and for its potential applications. My guess is that many microbiologists, like myself until recently, have only a hazy notion of it.

Let me set this in context. Bacteria have evolved a panoply of tactics for communicating both socially and antisocially with most anyone, be they other bacteria or cells of their host. For this they deploy small molecules (active only above a critical threshold concentration, hence quorum sensing), they construct pili to translocate DNA, and they export proteins via more than a half dozen secretory systems. Each of these tactics requires its own mechanics and makes its own demand for energy. Secreting proteins freely into the environment is particularly wasteful of resources because few of the molecules are likely to reach their intended target. To improve the odds, some bacteria depend on making direct contact between donors and recipients before exporting. Others parcel out the molecules inside specialized delivery structures—the subject of this post.

Figure1
Fig. 1. OMVs on an enterotoxigenic E. coli recovered from mouse small intestines 2h after intragastric inoculation. EM by Amanda McBroom. Source.

Vesicles On The March

Many, possibly most of the Gram-negative bacteria produce extracellular vesicles from their outer membrane (OM). These blebs, usually called outer membrane vesicles (OMVs), are spherical structures typically 20 to 200 nm in diameter formed by the outward protrusion of small bits of the OM that then pinch off to release the vesicles into the environment. Thus formed, the OMVs are bounded by a double-layered membrane with the usual OM protein, lipid, and lipopolysaccharide (LPS) constituents, and carry within them proteins from the periplasm. While details of the mechanism of vesiculation are yet to be elucidated, here’s one exciting example: The Pseudomonas aeruginosa signaling molecule PQS (2-heptyl-3-hydroxy-4-quinolone) stimulates OMV formation, apparently by inducing membrane curvature through interaction with the LPS.

Continue reading "Vesiculation: Another Bacterial Skill" »

August 12, 2013

Pictures Considered # 7. Cocci Divide at The Equator

by Elio

In 1962 Cole and Hahn published in Science an unassuming sounding paper entitled Cell wall replication in Streptococcus pyogenes. The authors asked the question: do strep cells synthesize their cell wall by intercalating new parts at different sites on their surface or does this take place at one site only? The question was not trivial. Cocci do not have a clearly defined mid-cell region as do rod cells. Furthermore, some rod-shaped bacteria were thought to use an intercalation mechanism for cell division. So the authors kept an open mind and laid out the following premise: If bacterial cell wall could be differentially labeled during growth to distinguish portions of different relative ages, answers to some of these questions might be forthcoming.

Figure1
Group A streptococci were grown with fluorescent-labeled antibody and transferred for 15 minutes to a medium with the same, non-fluorescent antibody. Source.

To begin, they made use of the fairly novel (well, actually “modern” would be a better term, as it was invented in 1942) development of fluorescent antibodies to differentially label portions of the wall during cell division. They used antibodies against the type-specific M-protein. The antibodies do not affect the growth of strep cells, so they can be used to track the synthesis of new cell wall in an actively growing culture. The authors used their antibodies in different ways, with the same results. In the example shown in the figure, they pre-labeled the cells with fluorescent antibody, washed them, then exposed them to non-florescent antibody for various times. Under the UV microscope, the original halves of the cell walls appeared as fluorescent “X’s”, which are in fact two back-to-back “C’s” representing the fluorescently-labeled older cell wall.

The authors’ concluded from this relatively simple and straightforward experiment that: ….in Streptococcus pyogenes, new cell wall is not diffusely intercalated with the old, but its formation is instead initiated equatorially along a circumference which is the site of the next cross-wall formation. Not only is this a definitive result but it provides an exciting picture, don't you think?

ResearchBlogging.org

Cole, R., & Hahn, J. (1962). Cell Wall Replication in Streptococcus pyogenes: Immunofluorescent methods applied during growth show that new wall is formed equatorially Science, 135 (3505), 722-724 DOI: 10.1126/science.135.3505.722

April 22, 2013

E. coli Cells Face FACS and Get Back into Shape

by Kimberly K. Busiek

Figure1
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.

Continue reading "E. coli Cells Face FACS and Get Back into Shape" »

April 18, 2013

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

by Elio

Figure1
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”?

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April 08, 2013

Holey Biofilm!

by Gemma Reguera

Figure1
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

Figure2
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!

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