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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March 27, 2014

Pictures Considered #15. The Dynamo at Work

by Christoph Weigel

ATPase_Fig1_half_300 Figure 1. Movie appr. in real time (i.e. depends also on your browser); actual rate without actin load ~100 rev/sec. Source.

Why on earth would one stare—even for a few seconds—at this blurry, low-res, pixelated movie (to the right), watching a jittery rod tumbling counterclockwise around its pivot? Because it's ATP synthase, a key enzyme of the energy metabolism in every living cell, that's rotating there. And when a molecule does its job by a rotational movement, well, then it takes a movie to demonstrate this. We turn thus to 'very small things considered': an enzymatically active ~530 kDa multi-subunit protein complex with a diameter of  ~9 nm and a length of ~20 nm. Biophysicists finally showed in 1997 what biochemists and crystallographers had suspected for quite a while but could not prove by their own experimental methodologies: ATP synthase—also known as FoF1-ATPase, or complex V of the respiratory chain—is a molecular dynamo. First a closer look into the screenplay for the movie before peeking briefly at the bigger picture...

Molecular tinkering, the professional way

Noji and colleagues assembled their object of interest in three separate steps, all standard lab procedures, by 'affinity coupling' of the individual components. First, they prepared F1 particles (with the subunit composition: α3β3γ) of ATP synthase with His-tags at the N-termini of the β subunits to affix them to Ni-NTA covered microscope slides and a biotin-tag at the γ subunit. Second, to construct an actin probe conjugated to a red-orange fluorescent dye they coupled ~1 µm-rods of polymerized and biotinylated actin to rhodamine phalloidine. Lastly they coupled the biotinylated actin rods to the biotinylated γ subunits of ATP synthase via streptavidin. The whole assembly is shown schematically in Figure 2. When infused with ATP in buffer solution, a small percentage of the molecular assemblies started rotating―in all cases counterclockwise―as seen under the epifluorescence microscope and filmed for Figure 1.

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February 03, 2014

Adhering To The 'Replicon Model' The Sloppy Way

by Christoph Weigel

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.

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December 09, 2013

Seeing How Antibiotics Work

by Elio

Figure1 Fig. 1. Seeing is Believing? Source.

One would assume offhand that the pathways for synthesis and assembly of the major constituents of a bacterial cell “talk to each other,” i. e. they are tightly interwoven processes. Tampering with the biosynthesis of one should affect all the others, right? Wouldn’t you expect, for instance, that if protein synthesis were to be suddenly stopped,nucleic acid synthesis would also stop and vice versa? Of course, in time this has to happen, but how fast? For a while at least, enzymes and ribosomes that present at time zero may well continue to churn out their products. Indeed, several major biosynthetic activities act as if they were independent of one another initially at least. (Not all, however. We know, for instance, that when bacteria are stressed, they undergo the stringent response wherein the synthesis of ribosomal and transfer RNAs ceases abruptly but that of mRNA continues.) The major biosynthetic functions of a bacterial cell act as if compartmentalized, so that inhibiting one does not necessarily result in the immediate inhibition of all others. It’s like when a car runs out of gas, it can still coast for a while. Thus, we must keep the time scale in mind. Over a short time range, say a couple of generation times, some of the biosynthetic activities continue and the cells become distinctive at a subcellular as well as a molecular level.

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September 30, 2013

E. coli Keeps Its Powder Dry

by S. Marvin Friedman

Figure 1. Left: Dictyostelium discoideum aggregating into a fruiting body. Source. Right: Adult Caenorhabditis elegans male. Source.

Whether inhabiting soil, fresh water, or marine ecosystems, bacteria are constantly facing the threat of numerous and effective predators such as protists, nematodes, or phages. To defend against such predation, bacteria have evolved a number of strategies, including getting larger in size, moving faster, producing defensive secondary metabolites, and forming biofilms. Because of its anthropocentric appeal, one strategy stands out—keeping a locked armory of genetic weapons. The strategy involves breaking open the armory when confronted by perils—activating the genes that were locked mostly to save the cost of expressing them when not needed.

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September 16, 2013

It’s Time for Salmonella

by Susan Golden

If one were to ask, “What happens when Salmonella infects the gut?” it might not be obvious that you’d also need to ask, “What time is it?” But indeed you’d need to know the latter to appropriately address the former. An invading Salmonella, intent on setting up camp, is trying to pitch its tent on a landscape that is changing throughout the day. The mammalian gut, like most eukaryotic and some prokaryotic cells, uses an internal timing device to control its physiological functions. The circadian, or 24-h (circa diem, about a day) clock employs an endogenous oscillator to control gene expression, metabolism, and other cellular processes in such a way that individual genes, pathways, and their components ebb and flow in activity with distinctive daily programs, meshing together to execute the enterprise of the cell. Among circadian regulated processes are aspects of the immune system, including components of the inflammatory response. Included are changes in the expression of several inflammation-related (“proinflammatory”) cytokines by macrophages not only in the animal but also ex-vivo. Not coincidentally, Salmonella thrives in the inflamed gut.

Bellet and colleagues set out to investigate the relevance of circadian rhythms of the mammalian immune response during infection by using a mouse model. In this protocol, animals are pretreated with streptomycin, overtly to rid the mouse of much of its intestinal microbiome (this is known as the “colitis model”). Pretreatment with the antibiotic changes the way the mice respond to Salmonella enterica serovar typhimurium, which would otherwise mount a systemic infection more similar to typhoid in humans. The handy mouse colitis model frees researchers from the restrictive and expensive need of using the other established model: cows. The mice pretreated with streptomycin, once infected with Salmonella, mount a massive inflammatory response in the gut, as do humans.

Figure 1. Representative images (10× magnification) of cecal inflammation in WT mice infected (Salmonella) or not at day or night. Source.

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August 19, 2013

A Whiff of Taxonomy – Archaeoglobus fulgidus

by Elio

Cells of A. fulgidus are typically irregular and with a dimple. Bar: 0.5 µm. Source.

Pick an archaeon, any archaeon, and you will find it has a story to tell. Not all archaea are exotic but plenty of them are. These stalwarts live in environments we humans call extreme, where they carry out what to us seem extreme types of metabolic conversions. Most have come rather late into our awareness. To redress their neglect, I picked one almost at random. It’s a hyperthermophile called Archaeoglobus fulgidus (the genus name derives from “ancient sphere,” the species from “shining,” for its UV fluorescent glow under 420 nm light). The genus was proposed by Stetter in 1988.

The family to which this species belongs, the Archaeoglobales, is typically hyperthermophilic. A. fulgidus grows optimally at 83 °C and is at home in hellish environments such as deep sea vents, oil deposits, and hot springs. A chemolithoautotroph, it reduces sulfate to sulfide, specifically to iron sulfide when given steel pipes to 'eat'. Since iron sulfide corrodes metal pipes, it is a nuisance to the humans tapping oil deposits to produce oil and gas. On the other hand, this skill may come in handy for detoxifying metal contaminants at high temperatures.

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April 29, 2013

The Art of Microbial Alchemy

by Gemma Reguera

“The Great Work of the Metal Lover” display, by Brown and Kashefi (Michigan State University) (top image). Included is the aesthetically-engaging, original glass chemostat used to grow the microbe, a metal manifold for gas distribution, and a heated glass copper column that removes any traces of oxygen from the gas supply. Cupriavidus biofilms and the gold grains they produce are visible at the bottom of the vessel (bottom image). Source.

In 2001, Kashefi and collaborators published an article in Applied and Environmental Microbiology reporting the surprising finding that several iron-reducing microbes can use gold as an electron acceptor for their respiration. These microbial alchemists included both mesophilic and thermophilic bacteria as well as hyperthermophilic archaea. The beauty of this process is that the oxidized form of gold provided to the microbes, Au(III), is soluble, whereas its reduced form, Au(0), is insoluble. Hence, the microbes respire soluble gold and precipitate it as gold nanoparticles on their outer surface plus, in the case of the Gram-negative bacteria, in the periplasmic space as well. These studies provided the first experimental evidence supporting the role of microbes in the formation of gold deposits in both hydrothermal and cooler environments, thus challenging the prevailing view that gold mineralization was an abiotic process.

Since then, the list of microbes with the ability to precipitate gold has grown. One of them, the Gram-negative bacterium Cupriavidus metallidurans, is an especially abundant component of the so-called gold nugget microbiota, i.e., the bacterial biofilms associated with gold grains. Elio covered this story previously in our blog. This bacterium grows at room temperature and its mechanism for gold biomineralization is one of the best characterized. For this reason, when artist Brown asked Kashefi, both at Michigan State University, to help him design an exhibit that allowed the audience to witness the process of microbial alchemy, Kashefi regarded Cupriavidus asthe microbe of choice.

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March 25, 2013

A Day in the Life: Eavesdropping on Marine Picoplankton

by Heather Maughan

ESP being tested by divers at the Monterey Bay Aquarium Research Institute. Source.

Observing microbes in nature is a challenge. Compared to what goes on in the lab, there is not much one can do with them out there. So, instead of bringing the bacteria to the lab, why not bring the lab to the bacteria? Imagine being able to capture the expression of genes of a community of microbes in situ, and over multiple time points. This movement of the microbial stage to natural environments has been done for microbial niches that are easily accessible, such as agricultural soil, hot springs, or mine washes. But inhospitable sites far from a lab, sites such as hydrothermal vents and the open ocean, pose a bigger problem.

The solution? A steadfast robot designed and dispatched by researchers at MIT and the Monterey Bay Aquarium Research Institute. Known as the Environmental Sample Processor (ESP), this robot gathers samples of seawater and stores them temporarily so as to preserve the RNA transcripts for subsequent retrieval and analysis. ESP is also able to perform DNA and protein hybridization to identify and quantify specific molecules.

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February 18, 2013

Bacterial Antidepressants: Avoiding Stationary Phase Stress

by S. Marvin Friedman

In bacterial quorum sensing, the absolute number of cells is irrelevant; only the number of bacteria in a given volume plays a role. (Credit: Copyright Wiley-VCH) Source.

High on the list of the exciting manners bacteria communicate with one another is quorum sensing (QS), a population-dependent gene regulation system that operates within a wide range of species. The general scheme of QS is as follows: at high population densities, signal molecules called autoinducers reach threshold levels, at which point they initiate a signal transduction pathway leading to transcription of specific genes. This altered gene expression allows the bacterial community to behave in a cooperative manner so as to achieve a common goal. In a sense, the bacterial population now functions like a multicellular organism. In many Proteobacteria, such as vibrios and pseudomonads, autoinducers are acyl-homoserine lactones (AHLs), compounds synthesized by the LuxI family of signal synthases and detected by the LuxR family of transcriptional regulators. Cellular activities regulated by AHLs include bioluminescence, biofilm formation, motility, and the making of virulence factors, among others. A large body of studies has illuminated the molecular mechanisms underlying QS, but identifying what’s in it for the species has not always been easy. In the paper to be discussed here, Goo and collaborators show how QS enables bacteria to avoid the perils of entry into stationary phase.

Death in the Stationary Phase

The researchers studied three closely-related species of the proteobacteria Burkholderia: the rice pathogen B. glumae, the opportunistic human pathogen B. pseudomallei, and the saprophyte, B. thailandensis. Each of these species has an N-octanoyl homoserine lactone (C8-HSL) signaling system. The basic experiment was to grow these three Burkholderia strains and their corresponding QS signal synthesis mutants in rich LB broth. (Elio pipes in: LB broth is actually a nutrient-scarce medium that supports exponential growth only at low bacterial density. Growth ceases soon, thus this medium may be an OK choice for studies of the stationary phase, all the more so because LB is scarcely buffered, which is relevant to this work. But for studies using growing cultures, LB is an abomination. For an erudite treatment of the limitations of LB medium, see here) The wild type strains survived long periods in stationary phase but the QS mutants died shortly after entering the stationary phase. These crashes were avoided by adding C8-HSL to the culture medium, indicating that QS was somehow involved in preventing death in the stationary phase.

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January 07, 2013

Living Wires of the Ocean Floor

by Gemma Reguera

In a recent post I shared with you how different microbes come together to breathe as one. In some cases, all it takes is the presence of conductive minerals such as magnetite to facilitate the exchange of metabolic electrons between two microbial partners. This allows the team to catalyze a redox reaction (for example, acetate oxidation coupled to nitrate reduction) that each organism could not have been able to achieve individually. The role of conductive minerals in such interspecies electron transfer is a good example of the diverse strategies that microorganisms have evolved to compete for the available electron acceptors, especially those with highest affinity for electrons (the most electropositive ones), as more energy is generated from the reactions.

The metabolic stratification of sediments reflects the reduction of the most energetic electron acceptor available for respiration: from oxygen reduction in the upper sediment region to sulfate (SO42-) reduction and then methanogenesis in the deeper layers. Modified from source.

In marine sediments, for example, oxygen diffuses from the oxic waters into the underlying sediment and is rapidly consumed by microorganisms in the upper region. This limits its availability as a terminal electron acceptor in the deeper layers, which are largely anoxic. As a result, microbes in the anoxic zone rely on alternative electron acceptors, choosing the most electropositive first. This also stratifies the anoxic region of the sediment, with each layer reflecting, from top to bottom, the sequential reduction of nitrate (NO3-), manganese (Mn[IV]), iron (Fe[III], which is often present as insoluble iron oxide minerals) and sulfate (SO4-2). Methanogens, which use the most electronegative electron acceptor (CO2), are usually active in the deepest stratum of the sediment. Interestingly, the reduction of sulfate generates hydrogen sulfide, a toxic gas with the chemical formula H2S. Sulfide production can be high in marine sediments, leading to a characteristic foul smell similar to that of rotten eggs. Unless removed, sulfide can expand from its layer into the oxic layer and poison the respiratory enzymes of oxygen-respiring cells.

Some microbes have evolved mechanisms to detoxify sulfide and/or use it as a substrate for growth. Sulfide can, for example, be oxidized to sulfate or other sulfur compounds by some species even in the presence of oxygen. It can also serve as electron donor for photosynthetic purple and green sulfur bacteria or for nitrate reducers. In addition, sulfide can react strongly with iron and manganese oxides to form iron-sulfur and manganese-sulfur minerals, respectively, which can also be used as terminal electron acceptors. The integration of all these microbial activities in the sediments helps prevent the expansion of the sulfide layer and keep it at millimeter or even centimeters away from the oxic layer. It also creates a harmonic metabolic balance among all the microbes in the sediments, so although each carries out its own reaction they can respond and adapt to sediment disturbances as a whole, integrated system.

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