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by J. Jeffrey Morris
 

I have a confession to make. I get stressed. A lot. I think I've spent the better part of the last three years with the vague feeling I'm forgetting something really important. I bet there are a few people in the reading audience that can sympathize – it seems to be a pretty common experience for as­sis­tant professors. The stressors come from all sides – pressure to secure grant funding, adminis­trative responsibilities, the constant stream of email, teaching and students, trying to find time to write that manuscript you've been putting off, and it all has to be balanced with the competing de­mands of your personal life. It all piles up on you in a big suffocating mound of stress and anxiety.
 

Or at least that's how I would have described it a few months ago. But a funny thing happened – around the first of the year, like many others, I decided to focus on my health and started a re­gu­lar workout routine. Four days a week, an hour of weights or cardio. And crazy thing, it was like the stress melted away. Nothing else changed – if anything, the time demands were made slightly worse by devoting four hours a week to a new pursuit – but despite the continuing pressures of academic life, I felt a lot better. Which sort of makes you wonder – was that long list of responsibi­lities actually the cause of my stress, or was it really all just because I was sitting down all day?
 

Bacteria get stressed, too. They obviously don't have to write papers or grants, but they do have to make a living in some pretty unpleasant places. Because they have limited ability to control where they are, they often find themselves in places where temperature, pH, or other environmental pa­rameters are outside of their optimal "happy zone." They also often find themselves confronted with antimicrobial chemicals that inhibit or attack critical points in their metabolism, like when we try to kill them with antibiotics.
 

When bacteria get overstressed, they often die. We've studied the mechanisms of death in great detail, and there are particular kinds of damage asso­ciated with different stressors. For instance, high temperature stress is associated with protein denaturation whereas osmotic stress is asso­ci­a­ted with membrane failure. Antibiotics usually have very specific molecular targets, and the most common drugs that kill bacteria (as opposed to just slowing them down) either inhibit the forma­tion of the cell wall causing the cell to explode, or else interfere with protein synthesis and create lethal mischief with the cell's metabolism.
 

Figure 1. A Assay design. Cultures were stres­sed, the stressor was removed, and cells were plated on agar containing/lacking agents that interfere with ROS accumulation; then cfu was determined. D Ampicillin-stimulated PSD. Wild-type strain 3001, treated with ampicillin (2.5 MIC) in the presence/absence of bipyridyl (0.4 MIC) plus thiourea (0.45 MIC) (BT). Cultures were plated on agar containing/lacking the bipyridyl-thiourea combination plus sucrose (BTS; n = 3). Source

But what if these stressed-out bacteria are like stressed-out professors, and these many apparent mechanisms of cell death are actually all manifestations of a single, sim­ple underyling cause? That's exactly the thesis that is pre­sented in a recent paper by Yuzhi Hong and co-authors. In this study, Escherichia coli cells received lethal expo­sure to a variety of different stressors: three antibiotics with completely different mechanisms of action, heat shock, and a mutation that prevented DNA synthesis at elevated temperature. At several time points following exposure, samples of cells were taken and the stressors were removed, either by thoroughly washing the cells to remove antibiotics, or by returning the cultures to lower temperature. As expected, when the cells were diluted and plated on regular agar, there was a clear decrease in the number of surviving cells as exposure times increa­sed. However, if the cells were instead plated on agar containing thiourea or catalase – reagents that remove reactive oxygen species (ROS) like hydrogen peroxide (H2O2) from the media – the lethality of all the stressors was reduced by at least an order of magnitude, and in some cases protection from ROS apparently eliminated the ability of the stress to kill the cells at all.
 

Hong et al.'s conclusion was that the main way that these stresses kill bacteria isn't the primary damage targets we usually look at, but rather a single, secondary stress – oxidative stress – that is initiated by the first stress. Oxidative stress occurs in metabolism when electrons are placed onto oxygen molecules instead of whatever compound they were supposed to go to, yielding ROS that can cause lethal modifications to other cellular molecules. Importantly, some ROS are free radicals that are able to create more radicals via chain reactions, explaining how ROS created by a primary stress can then amplify themselves and continue to damage the cell even after the first stress is removed. It's clear that the primary stress also damages the cells, because when Hong et al. expo­sed mutant E. coli that lacked the genes for repairing the injuries caused by the primary stressor (for example, DNA strand breaks), removing ROS wasn't able to save the cells any more. But it's also clear that ROS accumulation after stress is a major factor in transforming damaged E. coli cells into dead ones, regardless of the nature of the primary stressor.
 

Hong's paper is the latest in a slow-motion debate in the literature dating back to a 2007 paper in Cell from Kohanski et al. showing evidence for accumulation of hydroxyl radicals – the most lethal of the ROS – in E. coli treated with diverse lethal bactericidal antibiotics, but not in cells treated with non-lethal bacteriostatic antibiotics. Kohanski et al.'s paper was instantly controversial and spawned a number of studies casting doubt on the premise that ROS were involved, much less the primary mechanism of killing by antibiotics. For instance, papers by Keren et al. and Liu and Imlay, published back-to-back in Science in 2013, raised a number of serious concerns with the ROS hy­pothesis, such as the fact that antibiotics are still lethal – and in some cases more lethal – in anae­ro­bic environments, where ROS cannot be formed due to the absence of oxygen.
 

Dropping your laptop in the bathtub is a sure-fire way to experience the power of loose elec­trons. (Artwork by Sarah J. Adkins)

Despite 12 years of back-and-forth, I doubt this debate will be resolved by this latest work by Hong and cowork­ers. Myself, while I admit that it has its problems, I like the ROS idea for two reasons. First, it neatly fits my oversim­pli­fied mental picture of the careful balance in metabo­lism between energy harvesting and stress. As a human who has grown up in a world powered by high-energy electrons channeled into carefully-constructed circuits to generate wonders (like the machine you're reading this blog on), I can't help but compare the energy-generating flow of electrons in cells to that in our technology. Like an electronic device, cellular metabolism requires the care­ful and extremely precise shuffling of electrons to achie­ve the system's goals. But this precise electron flow is vulnerable to basically any mechanical per­turbation of the system – whether hitting a computer with a hammer, or mangling cell envelopes or proteins with antibiotics – and once the electrons get out of their proper channels, havoc en­sues. Imagine dropping your computer into a bathtub you're sitting in (Fig. 2) – when the physical mechanisms of cell or computer are taxed, electrons "leak" into places they aren't supposed to, leading to damage. Ouch!
 

The second reason I like the idea that ROS are an important component of antibiotic lethality is that I've seen similar things in my own work, in a completely different system. My lab studies the marine cyanobacterium Prochlorococcus, which we've shown has to live around other bacteria because it doesn't have the ability to remove ROS from the environment by itself. Under normal atmospheric conditions, there are enough ROS in the seawater where Prochlorococcus lives to kill it if it didn't have these bacterial "helpers" around. We did some experiments that showed that ROS removal was solely sufficient to explain this "helping" effect (Morris et al., 2011), but it's interesting to note that the presence of bacteria also protects Prochlorococcus from every other stress that we and others have thought to look at. There are published studies showing that bacteria protect Pro­chlorococcus from heat, cold, ocean acidification, and light starvation (Ma et al., 2018; Coe et al., 2016; Hennon et al., 2018), but anecdotally, I can tell you that bacteria also protect Prochlorococcus from high light, high pH, changes in salinity, temperature shifts even within their "happy zone", and even subtle differences between batches of culture media. Do all of these conditions lead to ROS production? Based on the "laptop in the bathtub" metaphor, maybe so. Prochlorococcus doesn't have many of the sensors for detecting environmental change found in other bacteria, so it is possible that all of these conditions can lead to relatively small primary damages that then yield self-amplifying and ultimately lethal oxidative stress in the absence of its bacterial helpers.
 

It's an interesting thing to think about. Obviously, if ROS are a major player in how antibio­tics kill bacteria, then it means blocking bacterial ROS defenses would be a good way to enhance antibio­tic therapies. But if all (or at least many) stresses are ultimately oxidative stresses, it will mean we need to focus more on the off-target electron transfers that occur in damaged cells. Are they all related to canonical electron carriers like NADH, as suggested by Kohanski back in 2007? Or are there other currently unknown mechanisms? There are so many possibilities, and I'm sitting here thinking about doing all of the experiments myself, and starting to feel a little stressed again – maybe it's time to stop blogging and get back to the gym…
 

References

Hong Y, Zeng J, Wang X, Drlica K, Zhao, X. (2019). Post-stress bacterial cell death mediated by re­active oxygen species. Proc Natl Acad Sci U S A, 116(20), 10064 – 10071. PMID 30948634

Kohanski MA, Dwyer DJ, Hayete B, Lawrence CA, Collins, JJ. (2007). A common mechanism of cel­lular death induced by bactericidal antibiotics. Cell, 130(5), 797 – 810. PMID 17803904

Liu Y, Imlay, JA. (2013). Cell Death from Antibiotics Without the Involvement of Reactive Oxygen Species. Science, 339(6124), 1210 – 1213. PMID 23471409

Keren I, Wu Y, Inocencio J, Mulcahy LR, Lewis K. (2013). Killing by Bactericidal Antibiotics Does Not Depend on Reactive Oxygen Species. Science, 339(6124), 1213 – 16. PMID 23471410

Morris JJ, Johnson ZI, Szul MJ, Keller M, Zinser ER. (2011). Dependence of the cyanobacterium Pro­chlorococcus on hydrogen peroxide scavenging microbes for growth at the ocean's surface. PLoS ONE, 6, e16805. PMID 21304826

Ma L, Calfee BC, Morris JJ, Johnson ZI, Zinser ER. (2018). Degradation of hydrogen peroxide at the ocean's surface: the influence of the microbial community on the realized thermal niche of Pro­chlorococcus. The ISME Journal, 12, 473 – 84. PMID 29087377

Coe A, Ghizzoni J, LeGault K, Biller S, Roggensack SE, Chisholm, SW. (2016). Survival of Prochlo­ro­coccus in extended darkness. Limnol Oceanogr, 61(4), 1375 – 1388. DOI doi.org/10.1002/lno.10302

Hennon GMM, Morris JJ, Haley ST, Zinser ER, Durrant AR, Entwistle E, Dokland T, Dyhrman ST. (2018). The impact of elevated CO2 on Prochlorococcus and microbial interactions with 'helper' bacterium Alteromonas. The ISME Journal, 12, 520 – 531. PMID 29087378

 
 

 


Jeff Morris is Assistant Professor at the University of Alabama, Birming­ham. His research interests are pretty much coterminous with those of Morrislab. He's also very interested in the history of the human species, and suspects that if he had it all to do over again he might have chosen to study molecu­lar anthropology instead of microbiology.

 
 

 
 

 

 

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Vincent, Michele, and Michael travel to San Diego to reminisce with Elio about his career, his work in microbiology, and his love for microbes and mushrooms.
 

Hosts: Vincent Racaniello, Elio Schaechter, Michele Swanson and Michael Schmidt

This Week in Microbiology - In the company of Elio - Episode 200! - YouTube

Right click to download TWiM #200 (44 MB .mp3, 61 minutes)

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Send your microbiology questions and comments to twim@microbe.tv

Music used on TWiM is composed and performed by Ronald Jenkees and used with permission.

 
 

 
 

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by Elio

 

A rather specific term, trophosome refers – as per the Wikipedia – to 'an organ found in some animals that houses symbiotic bacteria that provide food for their host.' Trophé (τροφή) denotes nourishment, soma (σῶμα), body, denoting that trophosomes are intimately involved in their host's nutrition.
 


Figure 1. The Giant Tube Worm, Riftia pachyptila and its Trophosome. Source. Frontispiece: Giant Riftia pachyptila in their habitat and abyssal fauna 2,630 m below the surface on the East Pacific Rise, during Oceanographic campaign Phare. Photo by Alf Håkon Hoel. Source

Animals that possess trophosomes include the giant tube worms of hydrothermal vents, Riftia pachyptila, and marine flatworms of the genus Paracatenula among others In the tube worms, the trophosome is seen as a greenish spongy organ located in the coelomic cavity (another Term of Biology for the 'body cavity between the body wall and the intestinal canal and other organs').
 

The trophosome of R. pachyptila  is composed of multiple lobules that contain bacteriocytes, that is, host cells filled with bacteria (see Figure). These as yet uncultivated gammaproteobacteria obtain energy by oxidizing hydrogen sulfide spewed from the hot vent fluid. The worms possess a hemoglobin that binds H2S and, via their circulatory system, delivers it to the bacteria in the trophosome. Oxygen present in the sea water is used as the hydrogen acceptor. The bacteria chemoautotrophically synthesize compounds such as NADPH and ATP that are used to reduce the carbon dioxide. The bacteria thus supply the host with needed organic compounds either by secretion or by being digested themselves.
 

In the tube worms, the bacteriocytes undergo a complex cycle, with mitosis and DNA synthesis restricted to the central part of the organ. In time, these cells are transported to the periphery, where they undergo apoptosis with digestion of the symbiotic bacteria. The reason for this 'cycle with terminal differentiation' is not yet known (We will come back to Paracatenula and its symbionts in a forthcoming post). But with all the transports of chemicals and the complex reactions that are going on within, why should trophosomes be anything but elaborate intricate structures?

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Small Things Considered by Christoph Weigel - 1w ago

by Elio
 

A Paradox, a Paradox, a Most Ingenious Paradox...
—Gilbert and Sullivan's Pirates of Penance
 

Tampering with the chemistry of our holiest of molecules may seem sacrilegious, but it is wide­spread and conspicuous. In fact, DNA modifications abound. Think, for starters, of DNA methyla­tion, which is the main tool of epigenetics, or an even longer known modification, the DNA gly­co­sylation that in some phages protects their DNA from degradation by the host. Such modifications play key roles in regulation and protection of the molecule yet, remarkably, do not impair the co­ding and base-pairing abilities of DNA.
 

Figure 1. Potential mechanism for oxygen-sulfur exchange, by activation of the phosphate group by alkylation, acylation, adenylation or phos­pho­rylation (R) and substitution with a nucleo­philic sulfur group. Source. Frontpage: Phos­pho­diester bond vs. phosphorothioate bond. Source

Just as disrespectful is the exchange of an oxygen in DNA for a sulfur atom in the phosphate backbone. You have now produced a phosphothiol, or, if you like it longer: phos­phorothioate. Note that sulfur and oxygen atoms have a kinship. Sulfur is just below oxygen in the periodic table and the two have similar electron configurations. Consequently, H2O and H2S are both used, albeit by to­tally different organisms, for autotrophic carbon fixation.
 

Phosphotiolated DNA (PT-DNA) is commonly found among bacteria and archaea. It was originally discovered in a species of Streptomyces, later in many others. This is sort of "Life imitating Art", as phosphothiolation (PT) was first developed artificially to sta­bilize oli­gonucleotides against nuclease activity. It was later found that this happens naturally and that bac­teria and archaea are capable of phosphothiolating (PT'ing). Diverse species can do it, including marine and soil-dwellers, pathogens and saprophytes, aerobes and anaerobes, and so on. As an example, about half the strains of pathogens such as Clostridioides difficile and Mycobacterium abs­cessus have this modification.
 

Figure 2. Proposed biochemical pathway of DNA PT modification. PT modification, con­fer­red by the DndACDE proteins, is predicted to start with the removal of sulfur from cysteine by DndA (Bottom left). The sulfur atom is then transferred to the target DNA site, which is cleaved and activated, likely by DndD and DndC, to complete the sulfur incorporation. Source

As an aside, the story of the discovery of PT-DNA is worth recounting. In the 1980's, Zhou el al. had a hard time ob­taining undegraded DNA preps from Streptomyces using agarose gel elec­tro­phoresis. They figured what caused it: it was due to the sensitivity of modified DNA to an acid formed in the anode from the Tris buffer used in electro­phoresis. Who would have thought? When they switched to HEPES buffer, the problem went away. In the process, they discovered DNA phosphothiolation.
 

Nowadays, PT modifications can be determined by the use of a highly sensitive LC-MS/MS technique that iden­tifies two-nucleotide sequences of PT-DNA. The pro­por­tion of bases that become so modified hovers around 5 per 10,000 nucleotides, depending on the species, which is quite infrequent as DNA modifications go. One recent analysis revealed that in Vibrio species, PT occurs in three distinct frequencies (2–3 per 103 nt, 3–8 per 104 nt, and 1–6 per 106 nt), the meaning of which is as yet obscure. However, it suggests that PT may play a role in restriction-modification, a point that is discussed in some detail in this paper.
 

The actual bases modified vary, usually being a a G or, more rarely, a C, or an A. Some species pre­fer G in GT pairs, others G in GA pairs, and yet others in both dinucleotides. The target sequen­ce of the DNA that is recognized differs among species. For example, it is GAAC in E. coli, GGCC in Streptomyces lividans, GGCC and CGGCCG in Vibrio. All are palindromes. In general, the level of mo­dification found is lower than the number of the corresponding sequences. A study of the ecolo­gi­cal distribution of the PT'd sequences of the genomes found in oceans tells that different PT con­texts may well vary with the habitat.
 

Figure 3. Correlation between PT sequence con­texts and Dnd protein sequences. Phylo­genetic analysis reveals a correlation between PT sequ­ence context and four of the Dnd pro­tein sequ­ences, and supports horizontal rather than ver­tical gene transfer for dnd genes. The PT sequ­ence context is color-coded as noted in the key. Source

In bacteria, PT'ing activity is encoded by a five gene cluster, dndA-E. One of its products, DndA is a cysteine desulfurase (an enzyme that removes the sulfur atom from cysteine) that assembles DndC into a DndCDE complex and endowes it with an iron-sulfur cluster (DndCDE-FeS). These proteins carry out the subsequent steps of cleaving the DNA and incorporating the sulfur atom. The process requires energy, which is supplied by ATP. It is worth noting that the Dnd cluster is carried on mobile elements as a genomic island, suggesting that it is likely to be transferred between species horizontal trans­fer (see Fig. 3).
 

Why PT'ing? What are its physiological functions? I pre­sen­ted above the suggestion that PT'ing may play a role in restriction-modification. Nevertheless, other data seems paradoxical. On the one hand, PT protects DNA from nucleases, which, incidentally, has the practical use to keep artificial antisense oligonucleotides from becoming digested in a host. But in vivo, PT renders Salmonella enterica DNA unstable after oxidative stress, that is, by the action of hypochlorite or H2O2 (keeping in mind that nucleases and oxidizing compound act very diffe­rent­ly). Sensitivity to oxidation agents is due to the changes in redox and nucleophilic properties of the DNA backbone.
 

But there is more to it. Upon PT'ing, catalase-deficient E. coli mutants become resistant to the ac­tion of H2O2, which may seem paradoxical. How to reconcile these two facts? Which is it, does PT enhance or decrease resistance to oxidation? A recent paper suggests an answer. The complex of the proteins DndCDE involved in PT'ing binds strongly to PT-DNA and, in the process, becomes a catalase that protects PT-DNA from damage by H2O2. All three proteins of the DndCDE complex are involved and depend on the iron-sulfur cluster in DndC, a point that is discussed in detail in the paper. This is a short-lived activity, as this Fe-S is susceptible to damage by H2O2. But it suffi­cient to keep the catalase mutant of E. coli going.
 

So, the conclusion is that PT does both. It makes PT-DNA susceptible from oxidative damage and, in a catalase mutant, it protects the DNA by the catalase ability of the proteins involved in PT'ing. It depends, therefore on the conditions and on the strain. But you've heard this before in the micro­bial world, no?
 

 
 

 
 

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Small Things Considered by Christoph Weigel - 3w ago

by the STC team
 

Foto by Davide Schiano

We are taking our customary two week vacation. Do you think we deserve it? Looking forward to seeing you again on July 8th !

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Small Things Considered by Moselio Schaechter - 3w ago


 
 
 


 

Why haven’t eukaryotes developed a CRISPR system? (the immune system doesn’t count)
 
 
 

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Small Things Considered by Christoph Weigel - 1M ago

by Elio
 

We resume our custom to list the posts from the last half year, lightly annotated.
 

Ecology

Source

Inside Earth, Microbes Approach  Immortality  Jennifer Frazer kindly let us reprint an article from her delightful blog "The Artful Amoeba". She talks about microbial life at great depths and with great depth.

A "Cultural" Renaissance  U. of Arizona's Paul Carini revisit "the great plate count anomaly" (that is, we don't know how to nurture most bacteria in nature) and proposes that we are in the midst of a significant upheaval in microbial ecology. But this is not without cost ("it's like driving a Ferrari from the list of its components," Paul says). He helps us think why and how to renew our interest in cultivation.
 

Evolution

Source

Go Ahead, Prove that Eukaryotic Cells Arose Via En­do­symbiosis  Take a yeast cell mutant that cannot grow on simple sugars and introduce into it an E. coli that can (but itself cannot make a required vitamin). Obviously, they can only grow if together. Is this an example of eukaryo­tic cells arising, Elio asks?

Happy Birthday LTEE!  It stands for E. Coli Long-Term Evolution Experiment, the Richard Lenski's lab 31-year-old which, as Roberto explains, has shed considerable light on our understanding of bacterial evolution.

The Complex and Mysterious Red Queen  Jamie borrows from Alice in Wonderland to explain how a worm (C. elegans) and pathogenic bacteria (Bacillus thuringensis) adapt to one another. Meet "recurrent selective sweeps" and "frequency dependent selection", concepts well worth learning about.

A Whiff of Taxonomy – Cycloclasticus  Here is a bacterium with rare talents for digesting poly­cyc­lic aromatic hydrocarbons. A related clade is a symbiont of deep-sea mussels but, alas, these can­not degrade PAHs. Christoph delves into this unusual and fascinating subject.

Genome with the Wind: CRISPRs reveal unknown dispersal mechanism  Jaime Zlamal, a post­doctoral researcher at La Jolla's Sanford Burnham Prebys Institute tells us of a strange finding: CRISPR sequences of widely geographically separated strains of Thermus bacteria are similar. How come? Did these sequences travel by air? If not, how?
 

Friends and Pathogens

Source

Sensing Quorum Backwards  As Elio  explains, maize beetles need to acquire a bacterial symbiont anew at every generation. The symbiont needs virulence factors to become established but later on, must turn them off. Quorum sensing is afoot.

Salamalga  Jamie toys with her new term to designate the intimate symbiosis between a sala­man­der and a green alga. It's harder on the algae than on the salamander, it turns out. Find out why and how.

Prokaryotic Pointillism: When a Sea Sponge Endosymbiont Paints the Bigger Picture  Our newest associate blogger, Janie Kim, deals with a bacterial endosymbiont of sponges that endows its host with protective chemicals in exchange for required amino acids and cofactors. Tit for tat.

Phage Therapy – an Update  The 100-year-old attempts to use phages to treat bacterial infec­tions have gained much currency now that bacterial resistance to antibiotics has become rampart. Elio discusses this challenging topic.

Dental Care for Your Pet: For Their Health and Yours  Canisius College pre-vet student Meag­han O'Neill considers the role of Capnocytophaga canimorsus in canine oral plaque and periodon­tal disease. She proposes investing in doggie toothbrushes.

The Fires of Saint Anthony – A Snippet  Ergot, an alkali-laden parasitic fungus of cereals, has wreaked havoc in the Middle Ages, casing burning sensations and madness. Roberto says that this is another example of microbes influencing human behavior.

Book Review: The Power of Plagues  Daniel reviews the 2nd Edition of The Power of Plagues by Irwin Sherman and concludes, not without some concerns, that it is "a fantastic read overall, with broad appeal."
 

Fungi

Source

How is Light Made? – A Snippet  The genes involved in making light are known for luminescent bacteria but, heretofore, not for fungi, Elio points out. The author of a paper that describes one such system say "…the fungal bioluminescent system presented here is a mo­le­cular playground holding numerous opportunities for basic and applied research."

Fungomania 1. Who's The Biggest?  A honey mushroom colony may be enormous but the lar­gest living organism it ain't. That honor goes to a stand of aspen trees.

Fungomania 2. Hyphal Comings and Goings  Hyphae are the conduits of water and solutes in mycelium, but, surprisingly, they don't all do the transporting in the same direction.

Fungomania 3. Farming and Alcohol  Fungus cultivating bark beetles use ethanol made by the decomposition of tree material to selectively inhibit undesirable fungal species.
 

Structures, Functions, and Regulations


Let's make some more tyrosine, together! (part 1)  Weevils nurture an endosymbiotic bacte­ri­um called Nardonella that, as Christoph explains, is good at making tyrosine. Why tyrosine? Well, this is a key component of the weevils' cuticles. But there is much more to this symbiont, which required penning a second part.

Nano Pétanques – A Snippet  Christoph examines the rules of the game involved in viral capsid assembly, among others.

Escape From the Wall  Roberto looks at fascinating examples of bacteria without wall, be they the venerable L-forms or the newly described S-cells formed when Streptomyces are stressed.

Blown Out of Proportion – The Role of ppGpp in Balanced Metabolism  Wash U.'s Petra Levin's grad student Elizabeth Mueller and postdoc Corey Westfall discuss its role in the stringent respon­se and beyond. What are the protein targets of "magic spot", as it was called by its discoverers? Read and find out.

The Pyrenoid – Not Your Average Cookie-Cutter Organelle  Janie introduces us to a major CO2-fixing organelle, the pyrenoid, that, surprisingly, is not surrounded by a lipid bilayer. Well worth becoming acquainted with.

The world of small RNAs and regulatory peptides in prokaryotes Microbiologist Matthias Gim­pel from Berlin's Technical University introduces this blog to the wonders of small regulatory RNAs and peptides and how one should think of them.

Risks and benefits of workouts while fasting (part 1)  This two-part account allows Christoph to jump into the complex physiology of "deeply starving" Bacillus subtilis and, thanks to environmen­tal metagenomics, to its relevance to the real world, especially to the endlessly fascinating peat bogs (part 2).

Extracellular Electron Transfer (EET) Goes Mainstream  Kevin Blake, a graduate student at Wash, U, St, Louis, tackles mineral respiration, the strange skill of some bacteria to "breathe" rocks (extracellular electron transfer). He focuses on a novel flavin-based mechanism of Legionella.
 

Viruses

Structure of chickenpox virus. They have a lipid envelope. Source

Distributed virusing?  Jamie strains our credibility by telling us of a plant virus (faba bean necro­tic stunt virus) that is made up of 8 segments, each of which is packaged in a capsid of its own. Beats the influenza virus, whose 8 segments reside in the same capsid.

Secondary Metabolites  UCSD graduate students Andy Bodnar, Michelle Prieto, and Carleen Vil­larreal tell us that secondary metabolite of Streptomyces acts as DNA intercalating agents to inhibit lambda phage replication. That's news.
 

Of Terms in Biology

Bacterial Fitness  A strain that can outgrow a relative is said to be more fit. Elio expounds on this.

Moonlighting  Not a bad term of Christoph's for multifunctionality, a widespread and exciting property of many bacterial enzymes.

Colicins, Bacteriocins, Pyocins and Tailocins  Roberto explains the history of the exciting uses for phage tails without the phage.
 

This and That

The Vicissitudes of a Talmudic Question  We are not always sure if a question is suitably Talmu­dic. We thought we should share with you an example of what goes on behind the scenes.

My Two Years in Copenhagen…  Elio reminisces about his two years as a postdoc in his favorite city.

Sydney Brenner (1927–2019)  A giant among the founders of molecular biology, Brenner also left us witty writings, an example here reprinted.

Geography and Geology  Elio is overjoyed about the width of the gulf between field biologists and bench experimentalists becoming reduced.

Chinese Surnames in Science  Elio points out that the astounding proliferation of research ar­ticles by Chinese authors poses a problem of unfamiliarity to Western ears (and vice versa). He proposes that learning something about features of Chinese surnames may help.

The Story of Free Use GFP (fuGFP)  Nick Coleman and Mark Somerville, Australian microbiolo­gists, wanted to use a superfolder GFP for their work but found that it had been patented. They got around it by modifying the protein and making it even brighter and... freely available to all comers.

Turdopoiesis  Elio toys with the charms of feces.
 

Talmudic Questions  

#161  Can you imagine a way in which the micro­bio­me is directly in­volved in human in­telli­gence and/or con­scious­ness?

#162  Can you imagine a eukaryotic virus that can infect a bacterium or an archaeon?

#163  Replication, transcription and translation achieve fidelity through hydrogen bonding at all stages except for the critical charging of tRNA with their cognate amino acid by dedicated amino acyl tRNA synthetases. Can you envision the evolution of a translation machinery that could ac­complish this latter step exclusively through hydrogen bonds?

#164  On Earth, L-amino acids in proteins go with D-ribo- or D-de­oxy­ri­bo-nuc­leo­tides. At which point in time during the evo­lu­tion of liv­ing sys­tems was the de­ci­sion made to make use of only one of the enan­tio­mer pairs?

#165  Describe the properties of a yet-to-arise new DOMAIN of life.
 

 
 

 
 

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Small Things Considered by Moselio Schaechter - 1M ago

by Elio

 

Here are books that cry out to be read. I start out with a list that appeared in a website called Five Books under The best books on Microbes. In an interview by Jo Marchant, reknown microbial eco­logist Paul Falkowski recommends these five books, some unanticipated: 

(Click to enlarge) Book covers (from left to right), Micrographia, Life on a Young Planet, Genesis of Germs, Plankton: Wonders of the Drifting World, Microcosmos.

  • The 1665 Micrographia by Robert Hooke, which Falkowski describes as the first scientific bestseller. He points out that "It was the first book that really showed the public what the world they could not see with their naked eye looked like."
  • Next is a Life on a Young Planet by Andrew Knoll, which tells the story of the first three billion years of life on Earth, all microbial. "He talks not just about how the evolution of life has been affected by the environment of the planet, but by how it, in turn, has changed the environment."
  • This is followed by a surprising choice, the 2007 Genesis of Germs by Alan Gillen, a creationist text that argues that microbes were created by God. A blurb by the publisher says: "germs are symptomatic of the literal Fall and Curse of creation as a result of man's sin, and the hope we have in the coming of Jesus Christ." This book is included here because of Falkow­ski‘s grave concern for the issue. As he says: "Once a religious worldview is embedded to displace scientific thought, it’s very difficult, even in children, to present data that changes that worldview."
  • The next selection is Plankton: Wonders of the Drifting World by Christian Sardet. As Falkowski says: "It’s an incredibly beautiful book, almost a coffee table book, but it’s also a serious science book. The beautiful photographs and colors are of organisms from the plankton that are photosynthetic — the phytoplankton — to zooplankton, to larvae of various animals in their planktonic phase."

  • His last choice is Microcosmos by Lynn Margulis and Dorion Sagan, engrossed proponents of mitochondria and chloroplasts being nanomachines derived from microbes some 3 billion years ago. Further, these authors champion the idea that evolution is not always about com­pe­tition and winners and losers, but also about cooperation.

I could not resist adding a few choices of my own.

(Click to enlarge) Book covers (from left to right), Guns, Germs, and Steel, March of the Microbes, Microbia, Thinking Like A Phage, Life at the Edge of Sight.

  • Highest on my list is Jared Diamond’s venerable and revered Guns, Germs, and Steel, which explains, among many key aspects of the development of humans societies, how a handful of germ-carrying Europeans could conquest huge empires in the Americas.

Next are some of my favorites, culled from a wide list of fine books written for the general public that point to the excitement of the microbial world. My choices, I hasten to add, are totally idiosyncratic.

  • March of the Microbes by John Ingraham leads us on an elegant tour through the microbial world, especially its aspects that that most readily concern humans (which is most of it). His subtitle "Sighting the Unseen" accurately portrays the scope of the book. It works.
  • Eugenia Bone’s Microbia starts out by sharing her experience about how a successful middle age writer on foods and mushrooms wanted to learn about microbes, especially those that dwell within larger organisms. She found out that she needed to go back and take a micro­bio­logy college course, which led to her writing this lovely book on how to see the world from the microbial point of view.

My last two choices are by friends and longstanding collaborators, especially in this blog. But I will maintain my composure.

  • Merry Youle’s Thinking Like A Phage is a most lively account of the vicissitudes of being the viruses that infect bacteria. But succeed they do, and they have become the most abundant biological entity on the planet. The text is illustrated with some phenomenal drawings. If you wish to become phage savvy (and everyone should!), this is the book for you.
  • Life at the Edge of Sight by Scott Chimileski and Roberto Kolter is, by their account, "A Photo­graphic Exploration of the Microbial World," and enormously successful it is. They blazed new trails, both in the content and in the illustrations, most of which are lusciously original. A tasty outlier, among books on microbes.

 

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Small Things Considered by Christoph Weigel - 1M ago
CRISPRs reveal unknown dispersal mechanism
 

by Jaime E. Zlamal
 

Ancestors of our cousins, the chimpanzees and bonobos, were once one single primate species which split into two when the Congo River formed about 2 million years ago. Not good swimmers, they are now very distinct species, with obvious physical and behavioral differences.
 

When there is geographical separation, we can anticipate genetic differences. That's exactly what a team led by Konstantin Severinov expected to find when they set out to study geographically dis­tant populations of Thermus bacteria. After all, these microbes generally live in hot springs at tem­peratures around 160 degrees Fahrenheit (~70° C). However, despite the communities existing a thousand miles away from each other in Northern and Southern Chile (El Tatio, Termas del Flaco), and across the world in Italy (Mount Vesuvius hot gravel and hot springs at Mount Etna), and Rus­sia (Kamchatka's Uzon caldera), in some ways individuals appeared remarkably similar. The study, published this year in the Philosophical Transactions of the Royal Society B, reveals a surprising re­semblance in some of the CRISPR spacers of differently-sourced Thermus strains.
 

A brief anatomy of a CRISPR
 

CRISPRs (clustered regularly interspaced short palindromic repeats) are short viral DNA sequences retained in the genome of bacterial cells that have survived infection by bacteriophages (phages). The CRISPR-Cas system includes these unique viral sequences – termed spacers – along with iden­tical repeats (between and flanking each spacer) and CRISPR-associated genes (shortened to the suffix "Cas").
 

CRISPR spacers act as a sort of adaptive immune response, allowing the progeny of the cell to avoid future infection by recognizing the same invading phages and dissecting their DNA. Multiple sequences from a phage can become spacers, and an organism can carry more than one spacer for a single phage. This helps prevent against viral mutation in one region rendering moot the be­nefit of that spacer.
 

Spacer sequences are arranged in a precise order, with new spacers added to the promotor end of the CRISPR array along with an additional repeat for each new spacer. Due to this consistent order of spacers, similarities between CRISPR arrays of different bacteria can be used to deduce their history and heredity. Over thirty subtypes of CRISPR-Cas systems exist, divided into two classes and six types. Since CRISPR arrays confer phage-resistance benefits, bacteria with useful spacers are expected to outcompete in the community among others that are not so endowed.
 

Crispy-hot spring CRISPRs
 

Diversity of CRISPR spacers in environmental Thermus samples. (b) Spacers from the same location are merged. The resulting diversity of unique 7877 spacers is shown in the circular dia­gram. Spacers from different locations that differ from each other by fewer than two nuc­leotides are connected by matching colour li­nes. Grey colour histograms on the outside show cluster size in log10 scale. For the more de­tailed legend see: Source. Frontpage: Elec­tron micrograph of an ultrathin section of T. ther­mophilus strain HB8. Cells were fixed in os­mium tetroxide, embedded in Epon, sec­tioned, and stained with lead citrate and uranyl ace­tate. The photograph was taken using a JEM lO­OU electron microscope at a magnification of 15,000×. The bar indicates 0.5 µm. Source

Severinov's team isolated phages from environmental samples and compared the viral sequences to corres­ponding spacers in Thermus CRISPR arrays from all sites. Strong correlations meant that these strains had been previously infected by local phages. Prior to this work, over 100 Thermus phages had been isolated, and eight complete genomes were available on GenBank. This study added five complete phage genomes to the list, three from Mount Vesuvius and two from El Tatio. When these five genomes were added to the reference data­base, the percentage of unique Thermus spacer matches to phage sequences nearly doubled, providing evidence that phages are under-sampled and under-studied in such contexts.
 

Before comparing CRISPRs in their samples, the team first sought out Thermus CRISPR spacer information in a publicly-available genomic database. Using a cutoff of two mismatches max, this preliminary analysis found 1567 unique spacers among Thermus isolate sequences. Many of the CRISPR spacers were distinct, as expected, and most of these were specific to the strain in which they were identified. Sometimes identical CRISPRs ap­pear in different organisms – not due to shared history but rather independent acquisition. This was the case with seven pairs of shared spacers found to overlap or in different types of CRISPR arrays. Of these Thermus spacers, 31 of them – or 2% – were found in more than one genome.
 

In contrast to the reference database, the diversity and number of CRISPR spacers identified in this study from environmental samples were much higher: 7246 unique Thermus spacers were recognized with only about 1% similarity to those in the database of sequenced Thermus isolates. Resampling of sites revealed spacer stability over time, with 37–49% of spacers shared between samples collected 27 months apart from Termas del Flaco, and 36–63% shared between samples collected 4 years apart at Mount Vesuvius. What truly stood out and surprised the team, however, was that spacers were common between remote sites.
 

To qualify as shared spacers, these sequences had to contain multiple identical spacers and re­peats in the same order in different samples ('identity' was defined as fewer than two nucleotide mismatches). Spacers shared between sites ranged from 405 spacers shared between two sites, to 78 shared in three, and even 4 spacers shared in four sites. Inexplicably, some Thermus spacers were shared between different continents.
 

Were these spacers acquired independently? This explanation appears the most likely, but the order and number of shared spacers precludes the chances that this occurred, especially for all of them. Two other thermophilic microbes, Sulfolobus solfataricus from Italy and Yellowstone (USA) and S. acidocaldarius from Japan and Yellowstone, have shown similar patterns of shared CRISPR spacers (56% and 95%, respectively).
 

So, what's happening? Is an airborne mechanism transporting DNA thousands of miles across earth and ocean alike, some sort of "air bridge" as the team suggests? Are these fastidious or­gan­isms hopping aboard phoenix-like birds and traveling from hot spring to hot spring? One thing is certain: Thermus bacteria are picky about where they live, and they sure aren't hiking from South America to Russia with a little holiday in Italy along the way. High homology between the newly annotated phage from this study and previously isolated phages (in some cases >80%) may con­found the results and assumptions based on spacer similarity, but multiple identical spacers de­veloping in the same order in Thermus from geographically-distant hot springs or hot gravel indi­cates something major. The team presumes that some extensive migratory pathway allows these bacteria and corresponding spacers to travel long distances. This still leaves questions unanswered. What do you think?
 

Reference

Lopatina A, Medvedeva S, Artamonova D, Kolesnik M, Sitnik V, Ispolatov Y, Severinov K. (2019). Na­tural diversity of CRISPR spacers of Thermus: evidence of local spacer acquisition and global spa­cer exchange. Philosophical Transactions of the Royal Society B, 374(1772), 20180092. PMID 30905­291

 

 





Jaime E. Zlamal is a postdoctoral researcher currently studying antibiotic re­sistance evolution at Sanford Burnham Prebys Medical Discovery Insti­tute in La Jolla, California..

  

  

  

  

  

  

  

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by Roberto
 

This is the tale of the tail, of a phage. Years before Alexander Fleming's chance discovery of penicillin, many scientists were already interested in bacteriolytic activities. Bacteriophages played a big role in the early days of searches for bacterial killers. Felix D'Herelle's 1917 discovery of bacteriophages was preceded by a report in 1896 by Ernest Hankin of a filtrable agent found in Ganges River water that killed Vibrio cholerae. Then, of course, there was Frederick Twort's 1915 publication describing a filtrable agent capable of lysing staphylococci. But Twort favored the explanation that this lytic activity was an enzyme and thus D'Herelle did not buy into the idea that bacteriophages were widely distributed. But a few years later, a lesser known player, André Gratia, became fascinated with bacteriolytic activities. Gratia put two and two together and nicely showed that Twort's activity was indeed due to a bacteriophage.
 

Gratia's interests in bacteriolytic activities led him to develop key rapid screening techniques for detecting them. Soon, he realized that there were not only bacteriophages, there were also proteins able to kill bacteria. In 1925, Gratia published the beautifully titled and stunning article: "Sur un remarquable exemple d’antagonisme entre deux souches de colibacille," wherein he describes "factor V," made by one strain of "colibacille" (nowadays known as Escherichia coli) and capable of killing another strain of the same species. Remarkable as this discovery was, it was not until 1946 that Pierre Fredericq, a student and long time collaborator of Gratia's, would come up with the term "colicin," to describe highly specific antibiotic substances made by some strains of E. coli capable of killing other strains of E. coli.
 

Figure 1. Early (1964) electron micrograph of pyocin (adapted from Source)

Other investigators became interested in colicin-like activities made by other bacteria. Quickly it became apparent that one could obtain such specific killing activities from many species. Funny thing was, in most cases, their synthesis was induced by the very same treatments that induced the production of bacteriophage from lysogens, for example UV light or DNA damaging compounds. This led none other than Francois Jacob (along with Lwoff, Siminovitch and Wollman) to propose, in an article entitled "Définition de quelques termes relatifs a la lysogénie," the term bacteriocin to describe bacteriolytic proteins such as the colicins whose synthesis, much like prophage induction, kills the bacterium producing it. They clearly saw a connection between bacteriolytic proteins and bacteriophage. But alas! Nowadays, the term bacteriocin is much more loosely used because we now know that there are many proteins made by bacteria with antibacterial activity but whose synthesis is not lethal. But, the story goes full circle because there is still a connection between some bacteriocins and bacteriophage…
 

Soon after defining the term bacteriocin, in 1954, Jacob discovered one such activity from Pseudomonas pyocyanea (now known as Pseudomonas aeruginosa). Following the bacteriocin naming convention, Jacob called this activity pyocin. Since then, several  pyocins have been described. But some of them, including the one originally discovered by Jacob, are particularly remarkable because they constitute that connection between bacteriocins and bacteriophage. As soon as people began purifying and characterizing pyocins it became clear that they were "weird." They were of extremely high molecular weight. But, most amazingly, when imaged under the electron microscope in the 1960s, they looked just like bacteriophage tails!
 

Eventually, it would be shown that pyocins were indeed phage tails, encoded from chromosomal gene clusters that were the remnants of prophages, now dedicated to synthesizing killer tails. For years they continued to be called pyocins but, as more and more examples from different species were identified, Jason Gill and Ry Young in 2011 proposed the term "tailocins" to describe the killer tails. The term seems to be catching on though not completely, pyocin is still widely used. Of course, this wonderful evolutionary co-opting of parts of a bacteriophage to yield a killing activity has not escaped our attention here at STC as Merry twice described the actions of tailocins in the past (see here and here). So, there you have it, the details of the tale of the killer phage tails through the trail of how they got their name. 

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