What may be the reason why E. coli is usually the most abundant facultative anaerobe in mammalian feces?
What may be the reason why E. coli is usually the most abundant facultative anaerobe in mammalian feces?
by Gemma Reguera
Smile (or not)!
Figure 1. One of our ancient relatives? Source.
After watching Hollywood movies of medieval knights with neat haircuts and bright smiles, it may shock you to be reminded that our dear medieval cousins looked anything but clean. The truth is that hygiene was not a top priority in the Middle Ages and germs were in heaven. This was a time in which cities lacked sewage systems and feces, urine, and garbage were dumped onto the streets or into the castle moat. Not surprisingly, outbreaks of water-borne diseases were a frequent occurrence. Add to that religious concerns about nudity, and it will not surprise you to know that even the most illustrious doctors recommended not taking baths regularly. As a result, everybody, from the lowest peasant to the most powerful king, stank like a smelly animal.
In this era of complete disregard for personal hygiene, people did pay some attention to dental hygiene. Why? Because toothache was a widespread malady and people would result to anything in their power to prevent it and alleviate it. They would rub on their teeth, for example, mixtures of fresh or burned scented herbs such as parsley, mint, and rosemary, and then rinse their mouths with solutions made of herbs, vinegar, wine, and/or alum (the latter is still use by many as a home remedy for sore throat). Alas, these practices were not effective enough to prevent dental bacteria from growing and causing cavities and infections. I also read that our medieval ancestors were advised to clean their teeth in the morning, rather than at night. I imagine this was done to ensure they had good (better?) breath. However, the morning practice left the oral microbes free to roam and proliferate throughout the long night hours. Hence, cavities and abscesses were very common and medieval barbers found a profitable side job: pulling out teeth!
Figure 2. Successive colonization of the tooth surface by commensal and pathogenic bacteria leads to the formation of dental plaque. Source.
A Mouthful Of Microbes
Would you care to know what grew on those medieval teeth? Luckily for us, dental remains archived in museums still contain oral bacteria preserved within the tartar deposits. Even with best dental hygiene practices of today, bacteria colonize our teeth and form multispecies biofilms (dental plaque). Streptococci and actinomyces commensals are often the first colonizers, recognizing specific receptor molecules in the salivary pellicle that coats our teeth. Other bacteria then join the initial film and all grow together while metabolizing the food debris that sticks to the biofilm matrix and soluble nutrients flowing through it. If allowed to grow thick, pathogens such as Streptococcus mutans and Porphyromonas gingivalis also join the biofilms. And that’s when the problems begin... S. mutants metabolizes sugars and excretes acid that destroys the enamel coating of our teeth and causes cavities. P. gingivalis, on the other hand, invades the epithelial cells of the areas of the gum supporting our teeth, leading to inflammation and some forms of periodontal disease. To make it worse, minerals from the saliva continuously deposit as calcium phosphate on the biofilms, creating a hard, rough surface that promotes more bacterial colonization while and also provides a tough armor for the dental plaque. These calcified biofilms is what we commonly know as tartar or dental calculus. Dental calculus traps the bacteria and food-derived matter on the teeth but also preserves them for long periods of time (many thousands years!).
Figure 3. Dental tartar from ancient teeth still contain dental plaque bacteria visible in electron micrographs (insert). The jaw/teeth remains date back to the late Iron Age/Roman times (Source), whereas the dental calculus shown in the inset micrograph is from 50,000-year old Neanderthal remains recovered from the El Sidron cave in the Asturian region of northern Spain. (Source).
The preservation of dental bacteria in ancient tartar is so good that you can still visualize the different cell morphologies in electron micrographs of even prehistoric dental samples (Fig. 3). Investigators from Australia wondered if the bacterial DNA was preserved as well and whether they could use our powerful DNA sequencing methods to profile the bacterial community in the ancient calculus samples. In a recent paper published in Nature Genetics, the Australian team reported an elegant and technically superb study in which they did exactly that. They managed to extract DNA from calculus collected from 18 medieval remains corresponding to both men and women living in rural or urban communities in Europe in the early (400-750 years ago) or late (850-1,000 years ago) Middle Ages. As a reference, they also sampled modern plaque and calculus and used the sequenced data available in the Human Oral Microbiome Database (HOMD) for comparison. Their approach was to generate and sequence PCR amplicon libraries of three hypervariable regions (V1, V3 and V6) of the bacterial 16S rRNA from the extracted DNA. The major challenge was to ensure that the sequences used for the analyses belonged in fact to the medieval dental plaque DNA and not to environmental DNA and/or modern DNA contamination. To do this, they followed strict procedures during sample collection, extraction, and DNA amplification steps. They also established authentication criteria based on comparisons to sequences generated from extraction blanks, ancient teeth, and environmental (freshwater, sediment, and soil) control samples. Such protocols lend credence to the results and are used in other paleomicrobiological studies.
Figure 4. Community composition of modern and ancient dental plaque (a) and frequency of the oral pathogens S. mutants and P. gingivalis in the samples (b). Source.
A Walk Through The Mouths Of Our Ancestors
To gain historical perspective of the evolution of dental plaque, the researchers analyzed dental calculus obtained from human remains from not only medieval times, but also the Bronze Age, and the Neolithic (early farmers) and Mesolithic (hunter gatherers) periods. A first glimpse at the phyla composition shows remarkable similarities in the community profiles of all the ancient samples (Fig. 4a). All contained the 15 phyla commonly found in the modern oral microbiome, with highest percentages corresponding to the Gram-positive phyla Firmicutes and Actinobacteria. These two phyla include commensal species critical to the formation and maturation of dental plaque. For example, streptococci (Firmicutes phylum) and actinomyces (Actinobacteria phylum) species are often the initial colonizers. The former are also the most abundant organisms in oral biofilms. But these groups also contain well-known oral pathogens (e.g. S. mutants, the leading causative agent of tooth decay).Thus, the researchers looked at the composition of each phylum in more detail, hoping to gain further insights into the microbial diversity of ancient and dental plaques and the frequency of pathogens.
In depth phylogenetic analyses revealed a shift in dental plaque composition from the Mesolithic to the Neolithic period. The dental plaque of the hunter-gathering Mesolithic humans (7,550–5,450 before present), whose diet was predominantly protein-based, were dominated by commensal bacteria in the Clostridiales and Ruminococcaceae groups associated with good oral health. By contrast, farming groups from the Neolithic (early farmers) and medieval groups contained both non-pathogenic (e.g., Clostridiales) as well as decay- and periodontal disease-associated taxa. The abundance of potentially cariogenic taxa was even greater in modern dental plaque. Potentially pathogenic groups were even more abundant in modern dental plaque. The higher frequency of cariogenic groups in human dental plaque since the inception of farming also correlated with a greater presence of the oral pathogens S. mutans and P. gingivalis (Fig. 4b). S. mutants sequences were not detected in the Mesolithic hunter-gatherers and early agriculturists of the Neolithic period (less chances of tooth decay!), but their frequency increased progressively since then, reaching highest numbers in modern humans. The periodontal pathogen P. gingivalis, on the other hand, was present in all the samples, but was more infrequent in the Mesolithic hunters than in all the farming groups. Skeletal evidence is also consistent with increases in tooth decay and periodontal disease upon the inception of carbohydrate-based diets in the farming groups. So there you have it: we (and our microbes) really are what we eat.
Less Sugar, More Brushing
The results paint a grim picture: our ancestors suffered (likely a lot!) from dental maladies but we are at a higher risk for oral disease than ever before. The farming for cereals in the Neolithic introduced soft carbohydrates in the diet and shifted the composition of the oral biofilms, enriching for more pathogens. With plenty of carbohydrates and inadequate dental practices, oral biofilms provided an ideal setting for the growth of pathogens throughout the Middle Ages. Dentistry and dental hygiene practices may have improved dramatically in modern times, but social inequalities still make dental care and preventive practices a luxury for many people. We also eat great quantities of processed sugars and our mouths now provide Las Vegas-style buffets for pathogens. Not only do we carry more pathogens in our mouths than ever before, our dental plaque is also less biodiverse. In ecological terms, less biodiversity means less productivity and less resilience against perturbations. Thus, our modern dental plaque is more vulnerable to dietary imbalances and/or invasion by less productive, potentially pathogenic species than our ancestors. Yet despite the fact that I harbor more pathogens in my mouth than my medieval great-aunt, I cannot but feel relieved to live in the 21st century, with toothbrush, toothpaste, dental floss, and regularly scheduled dental cleanings at my disposal. I shall remember to hug my dentist on my next visit.
Gemma is associate professor in the Department of Microbiology and Molecular Genetics, Michigan State University and an Associate Blogger at STC.
Adler CJ, Dobney K, Weyrich LS, Kaidonis J, Walker AW, Haak W, Bradshaw CJ, Townsend G, Sołtysiak A, Alt KW, Parkhill J, & Cooper A (2013). Sequencing ancient calcified dental plaque shows changes in oral microbiota with dietary shifts of the Neolithic and Industrial revolutions. Nature genetics, 45 (4) PMID: 23416520
Vincent, Michael, and Michele discuss how soil-dwelling bacteria induce the formation of root nodules on legumes via a protein called CYCLOPS.
Right click to download TWiM #73 (57.5 MB .mp3, 80 minutes).
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Figure 1. Source.
In 1956 I joined Ole Maaløe’s laboratory in Copenhagen for a two year postdoc. We worked on the connection between the rate of growth of Salmonella and its macromolecular composition, arriving at the conclusion that there was indeed a simple linear correlation between the cells’ nucleic acid and protein content and how fast they were growing. In trying to interpret this, Ole was influenced by the experiments coming from the labs of quite a few people, showing that the synthesis of many biosynthetic enzymes becomes repressed when the end product of their pathway is added to the medium. If, say, arginine is added to a culture, the enzymes involved in arginine biosynthesis will not be made. If so, in cultures grown in a rich broth, many biosynthetic genes should indeed be silenced. These should include all the operons for amino acid biosynthesis and for other building blocks present in this medium.
Figure 2. Ole Maaløe. Source.
Ole reckoned that if he kept a culture growing steadily for a long time in rich broth, the cells might shed some of biosynthetic genes because they would not be needed under these conditions. So he did the following experiment: He inoculated a large flask containing perhaps 3 or 4 liters of a very rich broth. As an aside, the kitchen at the State Serum Institute where Ole worked until he became professor at the University of Copenhagen was known for making exquisitely rich media. The ladies who worked there prepared their own meat infusion using the best Danish veal meat in the market and added to it carefully selected batches of peptone. Their broth allowed Salmonella to growth at a superfast 16 minute doubling time (try that with dehydrated commercial media!) So, you can expect that the cultures in such a superrich medium might be super-repressed.
Ole’s inoculum was small enough that the culture was still in exponential growth by the time it was time to go home. I ‘m quite sure that in order to slow down the growth he kept the culture at a temperature lower than the 37° optimum (but don't remember what it was). Before leaving the lab, he inoculated a second flask using a small aliquot from the first flask. The next morning he did the same thing, and he kept up this series of twice-a-day inoculations for (I think) one week. By the end of this time, the culture had been growing exponentially for perhaps 500 generations. He reasoned that this careful protocol was necessary to avoid the genes reawakening in the stationary phase, where they may be needed. Thus even an incipient entry into stationary phase had to be studiously avoided.
I don't recall the size of the inoculum he used for each transfer. Note that this is a crucial point because this step stacks the experiment against itself by creating a population bottleneck (a sudden decrease in population size).
At the end of the experiment, Ole plated the cultures on a large number of Petri dishes with agar made with the same broth. He then replica plated a large number of colonies (let me guess a total of 10,000) onto a minimal medium agar plates containing glucose as the sole carbon source. He expected to find that some colonies from the broth plates would not grow on the minimal medium. These would represent auxotroph, cells that had lost biosynthetic genes due to some mutation or other, preferably a deletion. To his chagrin, he found no such colonies. In conclusion, he had laid waste to a lot of broth and Petri dishes, but at least got his answer in one week.
I have wondered about this experiment in the intervening years. Of course, many in vitro evolution experiments have been carried out since, but best I know, none using this particular protocol. So, over 50 years later, what might be the reason for this failure? I can come up with a few possibilities that I’ll list below, but invite everyone to contribute their thoughts.
I enlisted the help of three people in the know. Here is what they had to say:
Kevin Foster, Professor of Evolutionary Biology, Department of Zoology, University of Oxford, pointed out that 500 generation is not a long time. He further suggested that “the minimal medium used gave the bugs glucose and there will be glucose and glycogen (which will be converted to glucose by the bugs) in meat broth. So the initial selection would have used the same enzymes as the final selection. To do this experiment again, I would recommend making the metabolic requirements non-overlapping. But it would also have to run for longer AND I would ahead of time, try to find a metabolic process that is known to be costly to run.” Furthermore: “auxotrophic mutants do not show much of a growth advantage if any at all (but we don’t work with these much)”.
John Ingraham, Professor emeritus, University of California at Davis, focused on the fact that for auxotrophs to become detectable, they would have to have a growth advantage over their prototrophic parents. “The growth advantage of an auxotroph would derive from its saving ATP (owing in the case of a deletion to making a tad less DNA, and in all blocking mutations by decreasing flow through an unneeded pathway below the perhaps residual flow escaping feedback inhibition, and making less of the unneeded enzyme than the level set by end-product repression.) and probably to a lesser extent saving carbon.” He then said that “Ole's medium is most probably one that would offer ATP and carbon excess. (Who knows what set its growth rate. but it supports a blazingly fast perhaps not improvable growth rate.) Instead, Ole should have used a minimal medium with a poor carbon source (for example succinate or lactate) In which ATP and carbon supply limit growth rate. Then end products that are not utilizable as sources of energy and carbon should be added to support growth of corresponding auxotrophs. These would include certain amino acids, uracil, purines, and certain vitamins. Such a medium, I would think, would offer a distinct growth advantage to auxotrophs.
Kevin Young, Professor, Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, calculated that the inoculum size must have been too small to contain any mutant that may have arisen. He posited, “I don’t think the experiment failed in the sense of disproving the original hypothesis (that auxotrophs would accumulate when grown in a rich medium). I’m just afraid that the repeated re-inoculation steps continually selected for the most prevalent cells in the culture, which were the un-mutated wild type cells.”
Elio adds: I agree that this last point is crucial and may suffice to explain the apparent failure. One way to repeat the experiment without this limitation could be to use a turbidostat, a device that allows constant growth at a maximal rate and a fixed population density. No evolutionary bottleneck here. This would overcome the major object of Kevin Y. and could be used to probe into the experimental situations suggested by Kevin F. and John I.
by Christoph Weigel
By good tradition, posts in the STC blog come with subtitles to let our readers have a (short) break to breathe deeply before diving into the next paragraph. The subtitles of this post come as—yes!—music titles, in a more oblique way referring to the content of the following paragraph and with the invitation to the readers to enjoy listening while having a break.
Take #1 - Caravan (Duke Ellington)
Figure 1. Transcriptional regulation in eukaryotes. Source.
In prokaryotes, it only takes a small jazz band to get the music grooving: piano and a rhythm section suffice. The promoter region of a gene is a tiny stage on which RNA polymerase (p) and few transcription factors (dr, b) improvise on a tune, i.e. they initiate or skip transcription. By contrast, it takes a big band in eukaryotes to perform Duke Ellington's 'Caravan'. And since not all the musicians fit to the narrow stage trombones and saxophones are placed elsewhere in the concert hall: transcription factors (tb, sax) bind to sites—enhancer or silencer elements—that are found upstream and downstream of genes and also within introns.
In its typical textbook style, figure 1 oversimplifies the complex interplay of RNA polymerase II with the various transcription factors at a eukaryotic promoter by reducing it to a two-dimensional array, suspiciously omitting downstream located 'gene regulatory sequences'. This flatters our human ability to perceive 2D-maps at a glance but it ain't got that swing. Admittedly, only a 3D-animation could catch the groove of the spatial intricacies that bring enhancer-bound transcription factors into the proximity necessary for the multiple protein-protein interactions that orchestrate eukaryotic transcription initiation. Let alone the possibility that transcription factors bound to one enhancer interact with protein complexes at two or more promoters simultaneously. Please note in figure 1 the stippled grey line tagged 'spacer DNA', we will come to this later.
Take #2 - Lonlon / Ravel's Bolero (Angélique Kidjo/Maurice Ravel)
Figure 2. DNA compaction stages in eukaryotic nuclei. Source.
Transcription from a eukaryotic promoter is not straightforward and only possible when DNA is locally relieved from chromosome compaction—it is virtually completely compact during mitosis—as shown schematically in figure 2. Look at this figure by starting with the lower part. The FISH technique made mapping of genes in/on whole chromosomes (1,400 nm scale) possible already three decades ago. More recent approaches allow dissecting chromosome territories in interphase chromosomes when transcription is active (700 nm scale, scratching at the 300 nm scale) and mapping of large-distance and interchromosome interactions by the 4C technique (chromosome conformation capture (3C)-on-chip) with a resolution down to the 300 nm scale or even lower.
Now look at figure 2 from the top part. 'Reading' DNA (2 nm scale) has become sort of trivial today and includes a detailed understanding of sequence-dependent variations in DNA curvature and helix stability. Mapping nucleosomes—DNA wrapped around histone cores—has left far behind the initially simple DNase technique. Covalent modifications at the N-termini of histones—acetylation, methylation, phosphorylation—trigger the 'tightness' of DNA-wrapping around nucleosomes and the density of nucleosomes on a given DNA stretch (11 – 30 nm scale). The degree of compaction is actively controlled as a means to regulate promoter activity as is further compaction by so-called 'scaffolding proteins' (30 – 300 nm scale). Modifications on one histone—or the lack thereof—greatly influence which modifications will decorate neighboring histone subunits in a nucleosome: there is crosstalk. Also, a number of enzymes modify both histones and non-histone substrates e.g. proteins involved in transcription and even ribosomal proteins that interact with rRNA. Because of the emergent complex syntax it seems appropriate to exchange the iconic term 'histone code' for the term 'histone language'. Last to mention here is the 30 – 300 nm scale where newly discovered long, non-coding RNA species, incRNAs, are involved in regulating the structure of chromatin together with a bunch of 'scaffolding proteins' as e.g. the SMC proteins.
There was this stippled grey line tagged 'spacer DNA' in figure 1, a somewhat vague abbreviation. The DNA sequences of these 'spacer' DNA stretches are not important for the functionality of the promoter but their three-dimensional organization—including their histone-wrapping and higher-order structures—certainly is. They have to bring the enhancer elements in proximity to the promoter so that the enhancer-bound transcription factors can play their part of the tune. Axel Visel and his coworkers addressed this topic indirectly in a genetic approach asking to which extent distant-acting enhancers influence the looks of mice (craniofacial morphology) during embryogenesis.
Take #3 - God Bless the Child (Billie Holiday/Keith Jarrett Trio)
Attanasio et al. prepared face tissue from 11.5 days-old mouse embryos and mapped active enhancers by ChIP-Seq analysis with the p300 enhancer-associated protein as target. Briefly, this is the order: prepare tissue and cross-link the p300 bound to its specific DNA binding sites, followed by chromatin preparation, sonication and antibody enrichment of the protein-DNA complexes (immunoprecipitation). Finally, sequence the recovered DNA fragments to detect active enhancers by virtue of the p300 binding sites (see here for details of the technique). These authors detected genome-wide 4400 putative distant-acting enhancers active in the craniofacial tissue (figure 3).
Attanasio et al. chose 205 craniofacial candidate enhancers for further detailed analysis by LacZ transgenesis. Briefly: PCR-amplified candidate enhancer sequences cloned into a LacZ reporter plasmid were injected into fertilized mouse eggs and implanted into pseudo-pregnant foster females. F0 embryos were collected at days 11.5, 13.5 or 15.5. Reporter activity was determined by LacZ-whole-embryo staining and only patterns observed in at least three different embryos were considered reproducible positive enhancers. From the 205 candidate sequences, 139 showed significant p300 binding in different parts of craniofacial tissue, 49 were extremely conserved and 114 were highly conserved in human (see example in figure 4). Please note the distance of enhancer hs746 from the Msx1 gene. To obtain quantitative data, they analyzed several enhancers including hs746 further by optical projection tomography (OPT) (figure 5).
Figure 5. Developmental activity patterns of enhancer hs746(Msx1). The in vivo activity of the enhancer was monitored at different stages of development (days 11.5, 13.5, and 15.5). The enhancer was reproducibly active in the craniofacial complex during embryonic development, with spatial changes in activity across stages. Side views are of LacZ-stained whole-mount embryos. Front views are optical projection tomography reconstructed 3D images. Regions of enhancer activity are shown in red. Detail from Fig. 5; Attanasio et al. (2013).
Having mapped distant enhancers of craniofacial genes and shown their tissue-specific activity during embryogenesis, Attanasio et al. turned to genetics. They tested, as a final step, whether three particular enhancers located in the proximity of functionally unrelated genes—again including hs746, ~235 kb(!) away from Msx1—are important in modulating craniofacial morphology. To this end, they created three separate mouse lines carrying deletion alleles for each of the three enhancers using a standard homologous recombination strategy for embryonic stem cells. Mice homozygous for each of the three enhancer deletions did not display gross craniofacial malformations or other obvious deficiencies. They measured the effect of each enhancer deletion on the expression of the presumptive target genes Snai2, Msx1, and Isl1 by quantitative reverse transcription polymerase chain reaction (qRT-PCR) for different craniofacial structures of individual wild-type and enhancer deletion embryos at days 11.5 and e13.5. They found a 1.5-fold down-regulation of Msx1 mRNA in Δhs746 (see figure 6). In this and the other two cases, the changes in transcript levels of the respective target gene were confined to tissue subregions in which the enhancer was active. Subsequently, they compared mouse skulls from wild-type and enhancer deletion mice at 8 weeks of age. They employed micro-computed tomography (micro-CT) to obtain accurate 3D measurements of the skulls. Three cohorts, each consisting of at least 30 mice homozygous for a deletion of one of the three enhancers, were compared with a cohort of 44 wild-type litter-mates. Statistically significant, Δhs746 results in a shortening of the face, a widening of the posterior neurocranium, a narrowing of the palate, and shortening of the cranial base. The authors noted that although both Δhs1431 and Δhs746 have significant effects on facial morphology in structures derived from regions with enhancer activity at days 11.5 and 13.5, there were also measurable changes in other parts of the skull, underpinning that tissue development involves crosstalk with neighboring tissue.
Figure 6. Expression phenotypes resulting from the craniofacial enhancer hs746 deletion. (left) In vivo activity pattern of hs746 (at day 13.5). OPT data are represented in red (LacZ, enhancer active) and green (no LacZ, enhancer inactive). (right) Expression levels of enhancer target gene Msx1 in craniofacial tissues dissected from wild-type (gray) and knockout (red) litter-mate embryos. Error bars show the variation among individuals of the same genotype; Mble, mandibular; Mx, maxillary; Nose, lateral nasal process. Detail from Fig. 6 B+D; Attanasio et al. (2013).
Attanasio et al. demonstrate that deletion of craniofacial enhancers can result in non-pathological but measurable changes in craniofacial morphology in mice. The authors conclude that isolated examples of sequence variants in distant-acting enhancers associated with craniofacial malformations—such as clefts of the lip or palate—have already been described in humans. Circumstantial evidence points to noncoding sequences, including enhancers, as contributing substantially to these processes. Pathological malformations may represent the extreme ends of the normal spectrum of variation observed in their study.
Take #4 - So What (Miles Davis)
It bothered biologists a lot in the 1950s when it became clear that genome size in eukaryotes did not correlate with organism complexity, in clear contrast to what was found for bacteria, archaea, and their viruses. Older readers might remember here the 'C-value paradox': "… the fact that a complex higher eukaryote such as Drosophila has a minute genome makes it impossible to argue that larger amounts of DNA are essential to carry out sequence-specific roles. In an organism with 100 times as much DNA as Drosophila, such as a salamander or a bean plant, most of the DNA could be unessential for coding or regulatory functions" (Gall, 1981). Although there were 'pre-modern' arguments for a chromatin-structuring function of non-coding DNA to enable transcription, the majority of biologists adopted the term 'junk DNA' as appropriate excuse to not care much about deciphering its role (see here for a summary). In their work Attanasio et al. do not explicitly enter the 'junk DNA' debate. They show conclusively, however, that experiments are the way—sotto voce: as always in science. The somewhat bumptious announcement by the ENCODE project to have solved (most) of the 'junk DNA issue' by presenting algorithm-derived predictions of function(s) for the major fraction of non-coding DNA deserves exactly the kind of experiments described by Attanasio et al.. Also, it's experiments that will put an (due) end to the decade-long intellectual sophistries on whether 'junk DNA' should be better called excess DNA, surplus, nonessential, or degenerate or silent DNA, garbage DNA, non-informational or nonsense DNA, worthless DNA, trivial DNA, vestigial DNA, redundant DNA, supplementary DNA, secondary DNA, or incidental DNA (see here for citations).
Take #5 (Encore) - Stop All That Jazz (Leon Russell)
After a talk, Nikolaus Rajewski answered in an anecdotal way a question from the audience how RNA interference (RNAi; see video here) was actually discovered: "Once upon a time when people got used to purify RNA by gradient centrifugation. They were excited to get reproducibly two distinct bands next to some smear in the upper part of the tube and lots of in the lower. The two fine bands turned out to be the large ribosomal RNAs and the smear in the upper part mRNA. The smear in the lower part of the tubes contained tRNAs, 5.8S RNA and huge amounts of degraded RNA—apparently junk—that they eagerly dumped into the sink. Some 20 years later, people stirred up that junk and found … a zoo of miRNAs and siRNAs". It apparently can be rewarding to dig in the junk for jewels. You may eventually find out why a mouse looks the way it does.
Christoph Weigel is lecturer at the Life Science Engineering faculty of HTW, Berlin’s University for Applied Sciences.
Attanasio C, Nord AS, Zhu Y, Blow MJ, Li Z, Liberton DK, Morrison H, Plajzer-Frick I, Holt A, Hosseini R, Phouanenavong S, Akiyama JA, Shoukry M, Afzal V, Rubin EM, FitzPatrick DR, Ren B, Hallgrímsson B, Pennacchio LA, & Visel A (2013). Fine tuning of craniofacial morphology by distant-acting enhancers. Science (New York, N.Y.), 342 (6157) PMID: 24159046
by Bernard Strauss
Figure 1. The Hopkins Martine Station in Pacific Grove, California. Source.
In 1949, at the end of my second year at Caltech the faculty seems to have decided that I really needed to learn some biology. The method the Caltech faculty adopted was to send me to the summer course in microbiology given at the Hopkins Marine Station of Stanford University by Cornelis van Niel.
The van Niel course consisted of a summer’s work of classes and laboratory. I was given an office, which I shared with Ruth Sager. The class would meet with van Niel for lecture and discussion and we would then do laboratory work based on the discussion. An idea of the course can be gained from the first exercise. The class suspended some bakers yeast in water and using a microscope looked at the suspension. Van Niel would then go around the room asking students one by one what they saw. Invariably the answer would be yeast cells and the process would continue until some student (possibly warned by those with experience from past years) would say "I see little circles." This was the answer van Niel wanted in order to illustrate the difference between observation and conclusion (I saw circles but van Niel was convinced I had been warned!).
Van Niel was a master at maneuvering class discussion. Again and again he would have the class "design" an experiment, making corrections and inserting controls and then when we were all finished and satisfied with "our" design, Wolf Vishniac, the TA, would wheel in carts with the equipment, media and strains to do exactly the experiment that “we” had designed. (Wolf was the son of the photographer, Roman Vishniac best known for his photographs of pre World War II Eastern Jews but also an accomplished biological photographer).
by Gemma Reguera
A Mouthful of Kids
In his second expedition to South America, Darwin discovered many new species of animals and plants. The field observations obtained throughout this 5-year expedition provided the intellectual framework for the maturation of his ideas on evolution. It also introduced the world to a tiny (2-3 cm in length) frog known as Darwin’s frog. The group includes the northern (Rhinoderma rufum) and the southern (Rhinoderma darwinii) species, which inhabit the central and southern forests of Chile (and adjacent areas of Argentina), respectively. As in many other amphibians, fecundation is external. However, Darwin’s frogs do not leave the fecundated eggs on the ground and exposed to environmental insults and predators. The males scoop them with their mouths and incubate them in their vocal sac. The dedicated dads feed their offspring after the eggs hatch, producing secretions analogous to milk that allow the tadpoles to grow in a protected environment, sometimes until they have fully developed into froglets. When the young are mature enough to fend for themselves, the male frog literally spits them out. You can see a short video describing this amazing reproductive strategy following this link. This behavior, generally known as neomelia, allows the male ‘surrogates’ to care for the eggs and then the young, maximizing survival throughout the critical tadpole stage. Unfortunately, deforestation in the regions inhabited by these frogs has resulted in vast habitat losses, leaving Darwin’s frogs in precarious conditions. The last sight of a northern Darwin frog was reported in 1980, leading researchers to suspect that this particular species went extinct years ago. The species has been tagged as ‘possibly extinct’. The southern species, R. darwinii, which has traditionally occupied a much larger region, has been able to survive, but population numbers have declined dramatically.
Vincent, Elio, Michael, and Michele review how microbial virulence can be increased as a consequence of community surveillance and adaptation to macrophages.
Right click to download TWiM #72 (53 MB .mp3, 74 minutes).
Send your microbiology questions and comments (email or mp3 file) to firstname.lastname@example.org, or call them in to 908-312-0760. You can also post articles that you would like us to discuss at microbeworld.org and tag them with twim.
Like other bloggers, I have a personal interest in visiting other blogs from time to time. I am glad to say that I am often surprised and pleased by the quality of offerings in the microbiological blogosphere. Here are a few of my favorite examples.
Scientific American sponsors a lot of blogs, several of which are of direct nterest to microbiologists. I greatly enjoy The Artful Amoeba, written by the gifted Jennifer Frazer, IMO, one of the very best science writers around. When she touches on microbial topics, beware! Both your senses and your mind will be set atingling. Her last post in this area answers the question that I, and I trust others, have had for ages, namely how can flagella of spirochetes and spirilla move their cell when they are encased in the periplasm. Get ready to think about how certain corkscrews (“cavatappi) work.
More about movement, this time of round bacteria. How does a round ball know which way to move? Lab Rat explains how the spherical forms of Serratia marcescens move about and how this differs in their rod-shaped configuration.
by S. Marvin Friedman
Figure 1. Persistence. Source.
The disconcerting ability of bacteria to evade death from treatment with antibiotics is achieved with two distinct strategies, resistance and tolerance. Resistance occurs by a variety of mechanisms, including drug efflux and/or preventing the drug from binding by modifying either the drug itself or its target. Tolerance, on the other hand, is a characteristic of a subpopulation of normal cells known as “persisters.” This phenomenon was first described by J. W. Bigger in 1944 who found that some members of a population of Staphylococcus aureus survived treatment with penicillin. Pathways leading to tolerance have been studied extensively in Escherichia coli, where they have been shown to be highly redundant and therefore hard to combat. At long last, the work of Lewis and collaborators suggests that a combination of antibiotics mat be the answer to this mode of bacterial defense.
Here’s how persisters arise: given that most antibiotics work on growing bacteria, slowing down their metabolism will make them insensitive to the drugs. Normally, a small proportion of cells in the population spontaneously enter a state of reversible dormancy. Commonly, this is thought to take place through the action of toxin-antitoxin modules. An example of such a mechanism relies on the activity of the HipA toxin, a kinase that phosphorylates a glutamyl-transfer RNA synthetase. This leads to the overproduction of the signaling molecule (p)ppGpp and the activation of the stringent response, which shuts down the synthesis of about 1/3 the genes of the bacterium and thereby leads to dormancy. But there are other dormancy pathways as well and their multiplicity makes finding a drug that inhibits the development of persistence a futile task. Thus, researchers have recently turned their attention to the search for agents that would “corrupt” a target in persistent S. aureus cells located within a biofilm.
Before discussing the paper in question, note that the theme of persistence has been often linked to that of biofilm formation. This is appealing because bacteria in biofilms often become insensitive to antibiotics, ergo can turn into persisters. Many chronic infections have been shown to involve biofilms, including osteomyelitis, endocarditis, deep-seated infections of soft tissues, gingivitis, infections of catheters and other indwelling devices. Particular attention has ben paid to the emergence of persisters in Pseudomonas aeruginosa biofilms.
This transmission EM of a thin section of a T2 phage infecting an E. coli cell was produced by Lee Simon in the late 1960’s. It is noteworthy for depicting a remarkably large number of properties of the phage and of the infection process. Can you figure out how many such characteristics are illustrated or at least strongly hinted at? I came up with nearly a dozen (see below). How about you?
Phage properties illustrated in this picture:
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.