Small Things Considered

The Spell of Coxiella

2017-07-10T05:00:00-07:00

by Elio

A few years ago, we celebrated one of the prettiest discoveries in modern microbiology, achieving the growth of Coxiella burnetii outside its host cells in laboratory media (see Coxiella Escapes from Cell!). The excitement was justified, as this organism had been a prototype of a strictly intracellular organism. Best I know, it does not naturally grow outside of cells, despite having the ability to do so.

Particularly inspiring is that cultivation in vitro was achieved by gazing at its genome sequence and thereby gaining a sense of its metabolic needs. A fairly straightforward medium containing amino acids and a particularly high concentration of cysteine along with a low oxygen tension (2.5% O2) did the trick (Figure 1). These host-free-cultured cells retain their virulence. By now, the use of axenic growth technique to grow C. burnetii has become commonplace.

Figure 1. Colonies of C. burnetii on the surface of agar after 14 days incubation in a 2.5% oxygen atmosphere. They are tiny, 0.05 – 0.1 mm in diameter. Source

A paper with the provocative title "Horizontally Acquired Biosynthesis Genes Boost Coxiella burnetii’s Physiology" appeared recently. Horizontal gene transfer (HGT) in a normally intracellular organism? Where did these genes come from? Did these sequestered organisms have conjugal visits? Not likely, so the acquisition of new genes must have happened before the coxiellae became prisoners. C. burnetii seems to be derived from its closest relatives, tick-associated coxiellae, which are benign endosymbionts that appear to contribute to the nutrition of their hosts. Antibiotic treatment makes the ticks less fit. The authors say: "…this corpus of data demonstrates that C. burnetii recently evolved from an inherited symbiont of ticks which succeeded in infecting vertebrate cells, likely by the acquisition of novel virulence factors." The inference is that HGT took place before that, most likely even before the organism became a symbiont of ticks.

It is widely believed that HGT is a major — probably the major — actor in prokaryotic evolution. We at STC have devoted quite a bit of space to it (See the post by Christoph Un Tour d'Horizon (1/3). But do not disdain the other two pieces of his trilogy — here and here). An advantage of studying HGT in coxiellae is that, like many endosymbionts, they have a reduced genome (C. burnetii has ~2 Mbp) and, due to selection against unneeded genes, most of the ones present can be expected to be consequential in the organism's particular environment.

The authors used stringent criteria to identify genes acquired by HGT. This is not easy because C. burnetii has few close relatives (Legionella and two other members of the Legionellales, all within the Gammaproteobacteria). Because of the uncertainties in the taxonomic positon of C. burnetii, the researchers excluded genes that may have come from close relatives. They were left with 172 genes that they identified with high confidence as being 'HGT-niks'. Curiously, none of these were carried in the plasmids of this organism. The identifiable genes have relatives in other bacteria, about half being found in members of the proteobacteria (alpha, beta, delta/epsilon), in keeping with the rule that HGT takes place more frequently among related species. These intriguing genes include some that encode for Type IV pili, structures that, for example, enable Acinetobacter baumanii to sop up DNA from the environment. Does this mean that C. burnetii used this mechanism for its HGT? Could be.

Some of the HGT genes are clustered, thus appear to have been transferred en bloc. The authors zeroed in on one particular cluster involved in the biosynthesis of LPS that appears to have been acquired in a single event, possibly from some other gammaproteobacterium. LPS is the best demonstrated virulence factor in this organism (there are many others by now, see here). So, this organism joins the long list of those whose bacterial virulence factors have been acquired by HGT.

The investigators found two other sets of genes, one set involved in fatty acid and biotin biosynthesis, the other in making heme. The fatty acid genes probably originated in a deltaproteobacterium or a spirochete. The arrangement of these genes is practically the same as in Spirocheta africana (again, suggesting that they were transferred simultaneously). In addition, C. burnetii obtained via HGT genes for pyruvate dehydrogenases, enzymes that convert pyruvate into acetyl-CoA (which is needed for fatty acid synthesis as well as ATP generation). C. burnetii also has two copies of the gene required for biotin biosynthesis, one being of HGT origin. Inhibiting the function of several of these genes decreased growth fitness in vitro.

Figure 2 Coxiella burnetii binds to macrophages through αvβ3 integrin, which triggers phagocytosis through an actin-dependent mechanism. The nascent Coxiella-containing vacuole (CCV) acidifies to pH ~5.4, which is characteristic of normal phagosomal development. By contrast to phagosomes, the CCV also acquires microtubule-associated protein light-chain 3 (LC3; an autophagosomal marker), a process that is dependent on bacterial protein synthesis. The nascent CCV develops through fusion and fission events with early endosomes and then late endosomes in concurrence with a further acidification to pH 5, which is characteristic of normal phagosomal development. Lysosomal enzymes, including cathepsin D (CTSD), start accumulating in the CCV by 2 hours after internalization, at which point the vacuole is at pH ~4.5. This pause in CCV development might allow conversion of the bacteria from small cell variants (SCVs) to the metabolically active large cell variants (LCVs). Between 8 hours and 2 days after internalization, the CCV expands to occupy an increasingly dominant portion of the cytoplasmic space of the host cell. RHO GTPase is likely to be involved in maintenance of the large vacuole, whereas the recruitment of RAB1B from the ER might facilitate the acquisition of additional membranes to create this spacious CCV. Source

The story of the heme genes has an interesting twist. I was not aware that regulation of heme biosynthesis involves a tRNA, namely tRNA^glu. It works this way: the first step in the synthesis of heme is catalyzed in many bacteria by glutamyl-tRNA reductase and glutamate semialdehyde aminotransferase, enzymes that convert glutamate into gamma-aminolevulinic acid, the universal precursor of heme (these enzymes and their connection to heme make for an interesting story in itself, see here). In C. burnetii, this tRNA is not used in protein synthesis but seems to be dedicated to heme production. Mutants in this C. burnetii tRNA^glu grow poorly (this organism cannot take up heme from the outside). Interestingly, this tRNA is not present in the non-pathogenic tick coxiellae. The authors suggest that heme biosynthesis could be a target for anti-coxiellal therapy.

Now, you may well ask, does this work further our understanding of the mechanism involved in Coxiella pathogenesis? Perhaps not directly, but it adds to the repertoire of things we know about this organism. As we wrote earlier, this bacterium is the agent of a somewhat rare disease called Q fever (Q is for 'query', even though Queensland is where it was first described). Q fever is a worldwide debilitating disease, something like a really bad flu, which affects both humans and domestic animals. It's highly contagious and highly infective — perhaps a single cell can cause disease. It is classified as a class B bioterrorism agent that has been weaponized and mass-produced under various biological warfare programs. Q fever is a zoonosis, acquired most often by inhalation from infected cattle and other domestic animals.

The most notable feature of C. burnetii pathogenesis is the bacterium's unique ability to survive and even thrive in acidic lysosomes, sites that are extremely inimical to all other known bacteria. One can say that C. burnetii breaks many of the rules for intracellular life. Within its vacuoles (known as Coxiella-Containing Vacuoles, or CCVs), the organisms proliferate to the extent that a CCV can nearly fill the cell. Quite a bit is known about the various steps in pathogenesis. The organisms are 'stealth pathogens' that avoid causing an inflammatory response (which could be lethal to them). See Figure 2 for details of the steps in pathogenesis. Notable is that coxiellal LPS is about 1000x less endotoxic than usual for this molecule but this doesn't mean that other of its activities are impaired, such as helping in evading the immune response and aiding in being taken up by cells. The organism's ability to evade an immune response is connected to the fact that they inhibit apoptosis.

Just how to tie together these facts to the HGT story is not yet clear, but it can be assumed with confidence that further progress will be made in the understanding how this unusual pathogen causes disease.

This topic was discussed on This Week in Microbiology Episode # 155 (below)

Talmudic Question #146

2017-07-06T04:00:00-07:00

Cross-section of a generic Gram-negative bacterial cell envelope. Lipid A comprises the hydrophobic membrane anchor of lipopolysaccharide (LPS). LPS is the major structural component of the outer leaflet of the outer membrane. Source

The Gram-negative outer membrane lipopolysaccharide (LPS) has three components, Lipid A, the core, and the O-antigen side-chain. The O-side chain and parts of the core can be deleted. In contrast, Lipid A appears to be essential. Why do you think that is?

This Week in Microbiology #154 & #155

2017-07-06T03:59:00-07:00

TWiM #154: Rigor, lotteries, and moonshots

At Microbe 2017 in New Orleans, the TWiM team speaks with Arturo Casadevall about his thoughts on the pathogenic potential of a microbe, rigorous science, funding by lottery, and moonshot science.

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

Guest: Arturo Casadevall

Links for this episode

Pathogenic potential of a microbe (mSphere)
Rigorous science (mBio)
Funding by lottery (mBio)
Moonshot science (mBio)

TWiM #155: Living in the stomach of a cell

Michele updates the TWiMers on Legionella in the Flint water supply, and Elio informs us about how horizontally acquired biosynthesis genes boost the physiology of Coxiella burnetii.

Links for this episode

Legionella in Flint water (The Scientist)
Q fever with Robert Heinzen (TWiM Special)
Horizontally acquired genes boost C. burnetii (Front Cell Inf Micro)
Image credit

Subscribe to TWiM (free) on iPhone, Android, RSS, or by email. You can also listen on your mobile device with the Microbeworld app.

Become a patron of TWiM.

Send your microbiology questions and comments (email or recorded audio) to twim@microbe.tv

When it comes to HERV-K, I beg to differ

2017-07-03T05:00:00-07:00

by Jamie Henzy

Although it's a regular occurrence for bacteria to acquire new genes whole cloth, by horizontal gene transfer, such events are rather rare for complex eukaryotes like us. Eukaryotes are vastly more inclined to take advantage of gene duplication events (see Christoph's series on gene generation tactics of prokaryotes and eukaryotes). An exception to this rule is the presence in vertebrate genomes of numerous retroviral sequences (endogenous retroviruses, or ERVs, for short) that were acquired presumably when a retrovirus infected a sperm or egg cell. Retroviruses make a double-stranded DNA copy of their genome and insert it into the host DNA to be replicated by the usual host replication machinery. One would imagine that an actively replicating retrovirus would interfere with the development of the fertilized egg, and it probably does more often than not. But sometimes the host can effectively silence the provirus (as the viral sequence is known post-insertion), or the provirus has a mutation that prevents its replication, and then the egg develops into a wee baby with half its mother's genes, half its father's genes, plus a wee bit more — a copy of the retrovirus in every nucleated cell of its body. Sounds like horizontal gene transfer to me.

What happens next? The broken or silenced ERV, freed from purifying selection, will begin accumulating mutations, hampering its potential for replication. During an initial honeymoon phase, however, it may replicate just enough to reinfect some new cells or copy itself into new genomic locations, or it can be copied by other retrotransposons such as LINE-1 elements, with the same results — copies in new locations. Eventually the sequence, behaving generally as a neutral allele, will be lost or fixed according to the whims of genetic drift. The amount of time that chance deliberates between fixation and loss varies with the length of the generation time. If we take the average generation time for humans to be twenty years (likely applicable for most human history, though it is lengthening in the present), then a neutral allele is expected to take ~800,000 years to reach fixation. During this unsettled phase, some individuals may carry an ERV where others have an unoccupied site. This state is known as insertional polymorphism.

Another insertionally polymorphic state for ERVs involves a sleight of hand based on a recombination event. When a retrovirus first inserts, the gene-coding region is flanked by long terminal repeats (LTRs) of several hundred to a thousand base pairs that are identical. These long regions of identity frequently recombine such that the intervening region is deleted and what is left is a so-called "solo LTR". In fact, the odds of this happening are as high as one hundred to one. Thus, the vast majority of ERVs in our genomes exist as solo LTRs. However, some individuals may carry a solo LTR where others harbor either an unoccupied site or a full-length ERV.

Figure 1. The three faces of ERVs. Top: an ERV first inserts as a provirus flanked by identical LTRs, shown by black hatched boxes. Middle: the LTRs can mediate recombination, resulting in the deletion of the virus genes and the generation of a "solo LTR". Bottom: if the ERV is still drifting in the population, some individuals may carry an empty site where others harbor an ERV. By: Jamie Henzy

As the LTRs acquire random mutations, they become less similar, making them less likely to mediate recombination. Thus, it's possible for an ERV to make it through this period of adolescent recklessness and survive as a full-length provirus. Regardless of whether LTR recombination occurs, mutations bombard the ERV sequences and, like meteors pockmarking the moon's surface, eventually pulverize the information content. So, the possible states of an ERV locus include three main forms: a full-length copy with two LTRs and coding sequence between; a solo-LTR; or an unoccupied site, all in various stages of sequence degeneration, including deletions that may shorten the sequences significantly (Figure 1).

Among the sixty plus families of ERVs found in the reference human genome, only one is known to have insertionally polymorphic loci: HERV-K(HML-2) ('HERV' for human ERV, 'K' for the tRNA^lys used to prime transcription, and 'HML' for Human MMTV-Like, reflecting the virus's relatedness to Mouse Mammary Tumor Virus). HERV-K began infecting the germline of our hominid ancestors ~35 million years ago, and hung in there, continuing to expand — either by reinfection or retrotransposition — even after the human-chimp divergence ~6 million years ago. This means that we share some HERV-K loci with chimps, but have others that are human-specific. More than 120 HERV-K insertions have become full citizens of human genomes, fixed in the population as a whole, so that every person carries them. However, at least a few copies of this family appear to be such recent arrivals that they are still drifting in the population, not yet having met their fate of fixation or deletion (green card holders?). This situation has led to the idea that HERV-K is mediating an ongoing, smoldering infection of the human genome even today.

However, the true extent of polymorphism has been difficult to assess. Why? Alleles at drift in a population vary in frequency, and the less frequent copies will go undetected unless a large enough sample is examined. You must pick through a whole heap of genomes to find them. However, copies that are not yet fixed are also the most recent, and therefore the most likely to still have functional elements — open reading frames (ORFs) from which proteins can be produced, or intact promoter regions that can interfere with transcription of nearby host genes. Therefore, these rare copies are the very ones that would be the most likely culprits if HERV-K were involved in disease.

Last year a group set out to hunt down these elusive, unfixed ERVs. Their haystack consisted of >2500 sequenced human genomes from the 1000 Genomes Project and the Human Genome Diversity Project. Sifting through this heap they spotted some previously unidentified rare insertions, plus some not-so-rare insertions that just happened to be missing among the handful of genomes that were pieced together for the human genome project. In fact, compared to the reference human genome sequence, they identified 36 "nonreference" HERV-K polymorphisms (remember, we're talking about insertional polymorphism) that ranged in frequency from <0.05% to >75% of the population. "What?" you ask. How did researchers previously miss copies that were in >75% of the population? Well, this group found empty sites in individuals at loci that are occupied by HERV-K insertions in the reference human genome, and were wrongly assumed to be fixed. In other words, their frequency is not 100% after all, but less.

Figure 2 (click to enlarge). Our kissing cousins. Source

Generally, African populations were found to have the highest frequencies of nonreference HERV-K2 alleles, and all of them but two were confirmed in these populations. This finding is consistent with the Out-of-Africa theory, whereby modern humans evolved in Africa ~200,000 years ago, with groups migrating outward 50,000 – 100,000 years ago. Migrating groups carried only a portion of overall human genetic diversity with them, and subgroups of these groups carried even less, creating an interesting decrease in diversity in populations according to their distance from Africa. Thus, Africa remains the most genetically diverse population, and their HERV-K alleles are no exception. Similarly, the presence of all but two HERV-K alleles in African populations indicates that these insertions occurred before subgroups began migrating outward.

Other HERV-K2 insertions were found to match those reported in Neanderthals and Denisovans — archaic humans with whom modern humans interbred after migrating from Africa. An outcome of such encounters is 2 – 3% Neanderthal sequences in the genomes of non-African populations, and as much as 5% Denisovan sequences in Pacific Islanders (Figure 2). So, were some of these HERV-K2 sequences acquired from our kissing cousins? Unlikely, since nearly all the insertions are also found in indigenous African populations, which did not interbreed with Neanderthals and Denisovans.

Figure 3. Top: representative full-length provirus showing LTRs (gray and black boxes) and virus genes (colors). Coding regions occur in different reading frames, represented by horizontal positions. Bottom: newly discovered intact provirus on X chromosome. The vertical lines represent base changes shared by other proviruses (black) or unique to this ERV (red). Adapted from Source

The group also found lurking within some X chromosomes a previously unreported full-length provirus, LTRs and all, with intact ORFs across all the viral genes (Figure 3). Only one other intact full-length ERV has been reported in the human genome. Their newly-discovered ERV is very rare, and most prevalent in African populations. Because LTRs are identical upon insertion, counting the mutations that differ between them and calibrating this to the mutation rate allows a rough estimate of time spent in the genome. By this neat trick, the ERV probably inserted sometime between 670,000 and 1.3 million years ago. With no apparent inactivating mutations, this provirus may very well be capable of replication. As the authors point out, even if it remains silenced by the host under normal conditions, disease states that interfere with silencing could result in its resurrection as an infectious retrovirus. Needless to say, this one is being looked at more closely in the lab!

The finding of an even higher level of insertional polymorphism than was previously known underscores how recently active the HERV-K family has been. In particular, some members may still be active, possibly with disease implications for the host that have yet to be discovered. The presence of these unfixed sites also highlights the importance of amassing ever larger databases of human genomes representing various populations. Who knows what rare, but possibly biologically important, ERVs are still lurking within the human population, or within your own cells as you read this!

Jamie teaches genomics and bioinformatics at Boston College, investigates ERVs, and is an Associate Blogger at STC.

References

Wildschutte JH, Williams ZH, Montesion M, Subramanian RP, Kidd JM, Coffin JM. Discovery of unfixed endogenous retrovirus insertions in diverse human populations. Proc Natl Acad Sci U S A. 2016 Apr 19;113(16):E2326-34. doi: 10.1073/pnas.1602336113. Epub 2016 Mar 21. PubMed PMID: 27001843; PubMed Central PMCID: PMC4843416.

Summer Vacation

2017-06-19T00:00:00-07:00

by the STC team

Foto by Davide Schiano

We are taking our customary two week vacation. Do you think we deserve it? See you July 3^rd.

News From the Missing Methanogenic Archaea Front

2017-06-15T00:14:00-07:00

by Elio

Figure 1. Whole cells of one of the hypersaline lake strains. Source. Frontpage: A twig from a hypersaline lake. Source

Best I can tell, methanogenesis remains a purview of the Archaea. Bacteria are still not known to do it and plants emit methane but apparently by recycling it from the atmosphere. We asked why this is in an early Talmudic Question (see here), which elicited some truly interesting comments. Some of which led Bill Martin into asking a whole raft of related questions, for example "How is it possible that methanogens require help from chemiosmosis in order to generate a proton gradient? Related: why don't they make acetate from H2 and CO₂ ?". Perhaps some of these questions have been answered by now and have graduated from the Talmudic class.

Figure 2. Thin section of a cell of one of the hypersaline lake strains. Source

But let me stop digressing. The news is that an international group of investigators have isolated novel methanogenic lineages of halophilic archaea. To find them, the researchers sampled hypersaline soda lakes and neutral lakes in southeastern Siberia and southern Russia. They found two kinds of methanogenic strains that are phylogenetically close to the halophilic archaea. One reason for the excitement is that halophilic archaea have been thought to come from the methanogens. The phylogeny of these new organisms adds to the plausibility of the notion.

Figure 3. Growth dynamics of strain AMET1 with MeOH + formate at 4 M total Na⁺, pH 9.5 and 50 °C Source

To isolate these strains, the workers grew samples in the presence of methane precursors such as a duo of formate and either methanol or trimethylamine, which are used in the methyl-reducing pathway, one of the three known for methanogenesis. They tried to imitate the provenance site by adding 4 M salt, using pH 7.0 for samples from neutral lakes and pH 9.5 – 10 for soda lake samples, and temperatures of 48 – 55°C. The cells are small motile cocci surrounded by a single layer cell wall. One unusual characteristic of these organisms is that they do not appear to protect themselves from the high osmotic pressure by storing organic osmolytes, but, rather, by accumulating a high intracellular potassium concentration.

The two isolates seem to have arisen early in the history of the archaea, as they belong to deep branches of the Euryarchaeota. They have been tentatively named the 'Methanonatroarchaea'. Details of a good many ecological (for example, salt adaptation), physiological, and comparative genomic facts are provided. The paper is unusual in that its 12 authors represent 6 different countries, with Eugene Koonin at the rear.

Retrospective, June 2017

2017-06-12T01:00:00-07:00

From the Library of Congress Shop (modified). Frontpage: Photo Credit Unhindered by Talent

Readers may be interested in learning that STC is being archived at the Library of Congress, thus its contents will be available in perpetuity (what will people make of it in the future? ). We are told that STC enjoys "active collection status." Its web archives are available here. STC has been also archived as part of the Wayback Machine (whatever that is ) and is available here. Don't worry, we don't feel immortal. Not yet.

Techniques And the Likes

Breaking the Oxygen Barrier in Microbial Cultivation Jesse McNichol, a postdoc at the Chinese University of Hong Kong, introduces us to a 'quasi-universal' medium’ that supports the growth of both aerobes and anaerobes. One of the tricks? Autocla ve the P and N sources separately, otherwise they make harmful ROS's. Jesse further discusses the interesting background to this story.

All Quiet on the Bacterial Front – The Evolution of Sterilization Techniques Ananya Sen, a graduate student at U. of Illinois at Urbana-Champaign delivers a romp through the ages on the many ways used to preserve foodstuff and sterilize material.

From Polenta to Peptone Ananya deals once again with microbiological techniques, this time the variety of media used to differentiate and select different bacteria. The title alone should make you wonder.

Pictures Considered #43. The Sinking of René Descartes? Christoph presents a new way to display data. Instead of a 2D graph, try a circle. It makes looking at big data downright pleasant.

Then and Now

Persistence Persists The life of our late friend Fred Neidhardt is celebrated by his erstwhile colleague Vic DeRita, who explains one of his less known contributions, this one on the physiology of salmonellae growing intracellularly. Fiendishly clever measurements were used.

The genome editing method CRISPR restored production of the protein dystrophin (light green) to muscle cells in mice with a mutation in its gene. Source

On the Discovery of CRISPR – An Interview Roberto interviewed the Spanish investigator Francisco Mojica, one of the discoverers of CRISPR. The story has some fascinating twists that deserve to be more widely known.

Why Did Jacques Monod Not Become Ole Maaløe A funny question, asked by Elio. Monod made gene expression understandable, Maaløe made sense of the physiology of bacterial growth. But Jaques Monod had started as a growth physiologist, so, why did he not follow up on that? Elio thinks he knows why.

Brudziński: On his Contribution to Early Bacteriology Piotr Polaczek, a microbiologist at Caltech, talks movingly about his great-grandfather, a Polish scientist who in the early days was deeply involved in what are now called probiotics.

The Ecology of Louis Pasteur Pasteur could be considered the earliest microbial ecologist, something that is overlooked. Elio explains briefly.

Structure and Function

Tales of Mystery and Imagination ...in two parts. Fellow blogger Daniel delves into the arcana of the Planctomycetes, especially with regards to their nonconformist cell envelopes plus a detour through their mode of chromosome replication – click here for #1, here for #2. Yes, Virginia, they do make peptidoglycan.

On Shape We reprinted a most enlightening article on the meaning of the multitude of bacterial shapes by Manuel Sánchez. It was first published in his blog, Curiosidades de la Microbiología.

Prions in bacteria Reprinted here is an article on the fairly recent finding of prions where one would not expect them. Vincent Racaniello wrote this for his Virology blog and we discussed it in episode 144 of the podcast 'This Week in Microbiology' (TWiM).

Fine Reading: Killing by Type VI Secretion Elio discusses a recent paper by McNally et al. entitled 'Killing by Type VI secretion drives genetic phase separation and correlates with increased cooperation.' Well worth knowing about.

Ecology

Feeding on Plastic Sabrina Fitzgerald, a student in Daniel's Canisius College, writes about a group of bacteria that are capable of degrading polyethylene. In view of the massive accumulation of plastics in the ocean, this just may be of some help.

Forget-me-not. CC BY 2.0 Chris Waits

Island Stories and Venus's Hair Christoph’s favorite islands are the Canaries, so it's not surprising that he takes on a tour of one of them, El Hierro. Not only does this one have a fascinating geological, biological, and even human history, but of recent it sports a somewhat unique underwater volcano that is covered by microbial mats called 'Venus’ hair'. The lady's mane, it turns out, is a a complex assemblage of many bacterial species that Christoph likens to dental plaque. Great microgeography!

Staying Safe in Space Fellow blogger Jennifer Tsang (see her at The Microbial Menagerie ) discusses possible microbial contamination in space and some of its possible consequences. Yes, it pays to be careful.

Relieving the Pressure: An Out of this World Discovery Mike Delmont, a graduate of Daniel's Canisius College, asks whether conditions on Mars could support 'methanogenic life'. The answer is a resounding maybe.

Genomics, Evolution, and Assorted Worries

Making Genes from Scratch? Eukaryotes do Christoph presents a trilogy on the question of the genesis of new gene (neogenogenesis?). After generously explaining what we mean by genes, he tells us how a new one arose in yeast and deals with some of its quirks.

Making Genes from Scratch? Prokaryotes not so much... Now, can you find novel genes in proks? Horizontal transfer provides new genes to a species, but the genes themselves are not new. So, do they make new genes from scratch? In proks, not that frequently, he says.

Making Genes from Scratch? Prokaryotes do too, after all Now that you think you know the answer, Christoph probes into the intricate choreography between bacteria and their prophages and concludes that new genes do happen here, albeit infrequently and over a long span of time. Such newly evolved genes better fit in the cell's physiology.

Do bacterial species really exist and why should we care? Kostas Konstantinidis and Roberto tackle this most intractable of subjects. Don't look here for an answer you can take to the bank, but relish in a lively consideration of how to think about it. It will help.

Un Tour d'Horizon (1/3) Everybody talks about horizontal gene transfer (HGT) but a critical treatment is highly desirable. Christoph discusses HGT frequency and consequences. To whet your appetite, here he discusses a paper that says: "at least 81±15% of the genes in each genome studied were involved in lateral gene transfer at some point in their history".

Un Tour d'Horizon (2/3) Along the Banks of the Helicase River is the subtitle of this post, wherein Christoph says that the E.coli helicase loader, operating in the earliest steps of DNA replication, has a phage origin, which seems like a bit of a surprise. HGT has wrought its deed at the heart of the bacterial cell.

Un Tour d'Horizon (3/3) The subtitle 'HGT as it Happens' tells you that it is not just a genomicist's plaything. It actually occurs in natural situations that are amenable to study. For a typical genome, one successful transfer takes place roughly every 7000 years.

Fungi and Other Euks

Fungi, The Tillers Fungi were likely involved in the colonization of land by plants, some 500 million years ago. They may have done so by stabilizing the soil.

Fungi © Moselio Schaechter

Making Like a Flower The preoccupation of fungi with ensuring that their spores are spread abroad is exemplified by their ability to change terminal leaves of some plants into what look like flowers. Pollinating insects are readily fooled.

Fine Reading: Unearthing the Roots of Ectomycorrhizal Symbioses Elio discusses an especially well-written primer on this subject of key importance to our biosphere.

Did the Neanderthals Eat Mushrooms? A recent report of DNA obtained from the tartar of Neanderthal teeth revealed some sequences corresponding to tiny mushrooms. For a couple of reasons, Elio isn't sure that proves that our cousins were mycophagists.

Meet the Xenophyophores! We reprinted a piece on these fascinating plate sized protists from a blog by Chris Mah, The Echioblog. They live in the depths of the ocean.

Books, books…

A Naturalist's Book of Viruses Fellow blogger Jamie Henzy reviews a book on viruses by M.J. Rossnink. She liked it.

Microbial Reading Roundup Daniel, an avid bibliophile, reviews six books on microbiological topics. Read on…

This and That

A Whiff of Taxonomy – Pseudoalteromonas We resurrect this section, which we have neglected recently. This genus is important in marine ecology and for making a raft of bioactive compounds.

Teach to Teach Elio muses about how little help he got when he started to teach and applauds current efforts to remedy that.

Symbioses

If You Were a Carpenter Ant Roberto discusses two papers on the 'summit disease', the urge of fungi-parasitized insects to climb aloft a plant. One is on the relevant metabolites made by fungi in this process, the other on the transcriptomics of the situation. Many things change, including an uptick in the transcription of many fungal biosynthetic genes and a downregulation of immune gene expression in the insect.

Screenshot from a YouTube video clip

A Snippet ‒ The Fluke of the Eye How parasites manipulate the behavior of their host is nothing short of astonish%shy;ing. Here is an example of a fluke in the eye of the rainbow trout. When the fluke has reached the right stage in its life cycle, it makes the fish easier to be eaten by birds, which is where its life cycle can be complete.

Sex and the Single Pillbug Elio loves wolbachias. Who doesn't? Except some of the infected insects, pillbugs here included. Feminization is the word. As Elio says:"To the adage that 'germs cause disease', we can add 'germs change sex.' Go tell that to your grandkids."

Microbe 2017

2017-06-08T17:00:00-07:00

by Elio

The annual meeting of the ASM, called Microbe, took place in New Orleans, June 1-5, 2017. I was there and not just for the crawfish etufée . A few notable changes from the customary routine were in evidence. For the second time, the Annual Meeting of the Society was held jointly with ICAAC’s (the more clinical arm of ASM). Although this certainly made for a larger meeting, I can't say that I felt the difference. Big is big, whether you're talking 5,000 attendees or 10,000 (not the exact figures). On the other hand, I can see the advantage of having people of contiguous interests meet under the same tent. And with the distinctions between the traditional branches of microbiology waning, in part but not only because of shared technologies, this is more than a marriage of convenience.

Kathleen Rubins was selected by NASA in 2009. Rubins completed her first spaceflight on Expedition 48/49, where she became the first person to sequence DNA in space. Source

This meeting was innovative in several ways. For pecuniary and other reasons, some sessions were held not in the traditional cavernous lecture rooms but in smaller open venues scattered in the exhibit hall. While this is primarily designed to draw more people to the exhibits, it had the pleasant effect of making it more cozy and informal. I gave a talk at such a site, on "tales from this blog" and enjoyed it thoroughly. I felt I was talking in a town square or something, almost touching the audience.

Also innovative was the display of plenary and other sessions on TV sets located strategically in the big hallway. No need to fight the distance of that gargantuan convention center (it's a mile long, no kidding. I recommend golf carts, which were nowhere in evidence).

In time, I'll report piecemeal here on the science I heard. Let me instead tell you of an interview at This Week in Virology with NASA astronaut, Kate Rubins. See here for her accomplishments. A Stanford Ph.D. in Biochemistry and Microbiology, she can claim something that made an appropriate splash. From her NASA website: "Dr. Rubins was the first person to sequence DNA in space, eventually sequencing over 2 billion base pairs of DNA during a series of experiments to analyze sequencing in microgravity. Dr. Rubins also grew heart cells (cardiomyocytes) in cell culture, and performed quantitative, real-time PCR and microbiome experiments in orbit."

What she sequenced was DNA from phages, bacteria, and mice with a portable DNA sequencer called MinOn. Not only that, in her almost 4 months in orbit she did two spacewalks totaling over 2 hours in length (they had nothing to do with her science experiments, but astronauts are called on to mind the store in addition to their specific tasks). Listening to her responses to Vincent Racaniello and Rich Condit's questions gave me a fittingly vicarious pleasure. Kate was generous in the extreme in the openness of her responses and her willingness to paint a vivid picture of what it's like to do science out there. I couldn't help asking her what astronauts in the Space Station do for fun. Well, they have to spend 2 ½ hour a day exercising to avoid bone loss. But what Kate did for entertainment was to appropriate a window of the spacecraft that pointed to the Earth and watch our orb for as long as she could. She described the experience in passionate terms, telling us how extraordinary an experience that is. She told us that the sight is truly gripping and that she could not have enough of it. Beats TV.

A nourishing meeting, all in all.

Press play to watch Kate Rubins on This Week in Virology.

front page image: Kate Rubins gets ready for one of the two spacewalks she performed during her mission.

TWiM #153: Covert pathogenesis

2017-06-08T04:58:00-07:00

The TWiM team ventures into preprint space with an analysis of type VI secretion across human gut microbiomes, and provide insight into urinary tract infection: how bladder exposure to a member of the vaginal microbiota triggers E. coli egress from latent reservoirs.

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

Right click to download TWiM#153 (40 MB .mp3, 57.5 minutes).

Subscribe to TWiM (free) on iPhone, Android, RSS, or by email. You can also listen on your mobile device with the Microbeworld app.

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Links for this episo

ASM Microbe 2017
TWiM Live from ASM Microbe 2017
Landscape of type VI secretion (BioRxiv)
Type VI secretion structure (jpg)
Activation of dormant E. coli in urinary tract infection (PLoS Path)
Image credit

Send your microbiology questions and comments (email or recorded audio) to twim@microbe.tv

Brudziński: On his Contribution to Early Bacteriology.

2017-06-05T01:48:00-07:00

by Piotr Polaczek

Józef Brudziński died December 18^th, 1917 at the age of 43. In his short life, he achieved prominence as a pediatrician, neurologist, bacteriologist, and also as a political figure. In Poland, he is remembered as the first rector of Warsaw University following its revival after a century of turmoil (partition of Poland, several uprisings, and World War I). He was also wholeheartedly involved in organizing several pediatric hospitals in Poland, at the time among the most modern in Europe. His work as a pediatrician on neurological signs in diagnosis of meningitis is widely known. Medical students all over the world are familiar with the tongue-twister, called Brudziński's sign or reflex, used in the diagnosis of meningitis. Less is known about his contributions to bacteriology and his seminal work on intestinal bacteria. One has to dig deep into the literature of the early 20^th century to gain insight into this equally important work. In online searches, the name Brudziński complicates matters, as the spelling of his name has many variations in the literature: Brudzińsky, Brudzinske, Brudsinski etc. The approaching hundredth anniversary of his death is an appropriate time to call attention to his largely forgotten early work that, along with that of a handful of others pioneers, forms the foundation of what we now call probiotic and microbiome research.

Józef Polikarp Brudziński (1874 – 1917). Source

Because in partitioned Poland, at the end of 19^th century there were limited opportunities to study medicine, Brudziński left the country and enrolled in the medical school programs in Dorpat, Estonia (now Tartu). The russification of the university and the expulsion of German faculty, however, led him to move to Moscow University. After graduation in 1897, he continued his education at St. Anna Children's Hospital at the University of Graz, Austria, under Theodor Escherich. Subsequently, he worked at l'Hôpital des Enfantes Malades in Paris with Jacques-Joseph Grancher, Antoine Marfan, and Victor Henri Hutinel. It is interesting to note that both Escherich and Grancher, a close collaborator of Louis Pasteur, are thought to be the first pediatricians for infectious diseases (Shulman et al. 2007).

At the end of 19^th century, bacteriologist as a profession, per se, did not exist, but a few scholars, mainly physicians, were engaged in investigations of the microflora of human gastrointestinal tract and its role in health and disease. This work may mark the beginnings of probiotic and microbiome research. Reminiscent of today, there was no shortage of hype, skepticism and controversies involved.

Theodor Escherich was the discoverer of Bacilllus coli commune, later renamed Escherichia coli in his honor. He is credited with carrying out the first systematic study of intestinal bacteria (1886). According to one of Escherich's assistants and later a renowned physician, Bela Schick, the bacterial era was inaugurated in pediatrics by Escherich, who had a longstanding interest in bacterial flora of the gastrointestinal tract. It was believed at the time that bacteria are responsible for decomposition of proteins to harmful putrefaction products that could be absorbed from the bowels into the blood stream, a form of auto-intoxication. Escherich suggested fighting this condition by the introduction of acid-producing bacteria, first described by Louis Pasteur (Pasteur 1858), and including carbohydrates to the diet based on a mutual antagonism between the saprophytic intestinal inhabitants and the acid-producing bacteria.

In Escherich's clinic, Brudziński was to test this hypothesis by performing experiments aimed at combating intestinal putrefaction in dyspeptic infants using the acid-producing Bacillus lactis aerogenenes (now Enterobacter aerogenes), which was known to ferment sugars with the formation of lactic acid and gas. This organism had previously been found by Escherich to be one of the two dominant bacteria in stools of healthy infants (the other being E. coli. Note that this was before knowing that the dominant intestinal bacteria are strict anaerobes). Brudziński first examined fetid stools of several dyspeptic infants and found that most grew out Proteus vulgaris. He then administered pure cultures of Bacillus lactis aerogenes, which proved successful. The foul smell of the stools subsided, and they regained their natural acidic smell. Proteus was now absent. A similar effect was achieved by feeding patients large amounts of milk and sugar. He concluded that the symptoms of auto-intoxication observed in dyspeptic children were due to absorption in the intestine of toxins derived from Proteus. Brudziński also performed experiments with animals. Proteus injected under the skin of mice was lethal, while no symptoms were observed if mixed with food of young dogs and kittens. To identify the source of Proteus found in the stools, Brudziński examined samples of raw and boiled milk for the presence of the bacteria. Proteus grew only in previously boiled milk, seldom in fresh milk, and never in acidic milk.

(Click to enlarge ) Escherich's clinic in Graz, Austria. Theodor Escherich (center), Meinhard von Pflauner examining a child, and Józef Brudziński (far left) with Ernst Moro inoculating a laboratory animal. Source

Research on bacterial microflora of the gastrointestinal tract was quite prolific at the time. Two newly discovered species of intestinal bacteria were added to the list. Ernst Moro discovered a gram-positive bacterium, Bacillus acidophilus (now Lactobacillus acidophilus ), claiming that it is a dominant species in breast-fed infants (Moro 1900). At the Pasteur Institute, Henri Tissier observed a Y-shaped bacterium and, because of its tendency to branch, named it Bacillus bifidus (now Bifidobacterium ) (Tissier 1900). Tissier disputed Moros' assertion of the dominance of B. acidophilus, claiming that B. bifidus is of primary importance. The controversy ended with Moro's own admission that B. bifidus is present in far greater numbers in breast-fed babies, and both agreed that B. acidophilus was predominant in stools of cow's milk fed babies.

Meanwhile, Ilya Metchnikoff, also working at the Pasteur Institute, turned his attention to the newly discovered Bacillus bulgaricus (now Lactobacillus delbruckii subsp. bulgaricus) (Grigoroff 1905, Massol 1905, Cohendy 1906). This species is not only a powerful acid producer but is also resistant to high acid concentrations. The large numbers of centenarians living in Bulgaria, where lactobacilli-containing yogurt was a dietary staple, convinced Metchnikoff that B. bulgaricus may provide the secret to longevity by replacing the toxin-generating bacteria in the colon. Whether this longevity may be attributed to the health benefits of yogurt or was simply a consequence of unreliable birth records is a matter of contention. Mechnikoff was not a physician and contributed little to experimental work, but was a staunch advocate of the health benefits of sour milk. This caused a media frenzy. The popular French daily, Le Matin, with front page headline: "Vive la Vie!" (Long live Life!), announced that Metchnikoff found an elixir of eternal youth (Vikhanski 2016). Subsequent studies with B.bulgaricus in other laboratories put into question whether it can survive in the stomach, much less colonize the colon. Metchnikoff's conviction, bordering on obsession, with the notion that acid producing bacteria may prolong life did not go unnoticed. As a result, he has been proclaimed the father of probiotics. His regimen of daily consumption of sour milk probably did not prolong his life by as many years as he had wished for. He died in 1916 at the age of 71. However, his accomplishments in immunology – notably, the discovery of phagocytosis, for which he was awarded the Nobel Prize in 1908 – earned him immortality.

The research exemplified above, relatively high-profile at the time, received recognition in the literatuVre, but was also met with a dose of skepticism, as demonstrated by the following:

"Recently, renewed attention has been called to the possibility of influencing intestinal fermentation and putrefaction by the administration of cultures of bacteria. An old method was to administer brewer's yeast. More recently Brudziński, Metchnikoff and Tissier have employed cultures of organisms that occasion lactic acid fermentation. The same applies to the administration of sour milk, buttermilk, koumiss, and similar preparations. The value of this form of treatment has not yet determined. It is doubtful whether the extravagant claims recently forward(ed) will be substantiated by further investigation." (Stengel 1908, p. 340)

The concept of influencing the composition of intestinal microflora with the use of mutually antagonistic bacteria was also discussed in terms of individual contributions. Alexander Poehl, a chemist and pharmacist from St. Petersburg was the first to discover that sour milk diminishes intestinal putrefactions. Only later were cultures of lactic-acid producing bacteria used in attempts to suppress this condition. Metchnikoff's contribution to researching the topic was questioned in another report:

"Poehl, Brudziński, Fischer, Rovighi and Embden then turned their attention to sour milk and found that that too would inhibit intestinal putrefaction to a certain extent at least. This work is in reality the foundation upon which Metchnikoff built his sour milk therapy. His claim to recognition appears to depend largely on the fact that he popularized this form of administration of lactose and lactic acid". (Kendall 1911, p.160)

Yale researchers Rettger and Cheplin provided a more detailed list of workers in the field:

Poehl (1887) noted that sour milk when ingested decreased intestinal putrefaction. This observation was confirmed by Rovighi (1892), Embden (1894), Brudsiński [sic] (1900) and Fischer (1903). Tissier and Martelly (1902) stated that the chief agent in effecting inhibition of putrefying bacteria is probably the lactic acid produced by the lactic acid bacilli. Tissier and Gasching (1903) found that acid-producing bacilli are able in a sugar-containing medium to arrest the growth of putrefactive organisms, thus confirming the conclusions of Bienstock (1899). (Rettger and Cheplin 1921, p. 5)

Thus, at the turn of 19^th and 20^th century, work on the bacterial gastrointestinal microflora thrived. This early fascination soon began to wane, but was kept alive for some time, thanks in part to sensational news with the promise of prolonging life, only to die with the advent of Fleming's "miracle cure" – penicillin. The early pioneers in the field are now largely forgotten. Today, as a consequence of antibiotics overuse and misuse and the growing threat of antibiotic resistant bacteria, we are witnessing the re-emergence of such studies, on a vastly larger scale by a rapid expansion of probiotic and microbiome research. Technological advances allow us to study human-inhabiting bacteria as a collection of numerous species and to analyze functional, complex interactions between the host and the microbiota, with new hope of preventing and treating a range of human diseases.

As mentioned above, Brudziński, apart from being a physician, was involved in other pursuits such as organization of several hospitals in Poland, launching of the first Polish pediatric journal, Przeglad Pediatryczny (Pediatric Review) in 1908, as well as actively participating in political life. In 1916, a declaration of Emperors Wilhelm II of Germany and Franz Joseph of Austria promised the creation of the Kingdom of Poland. This was regarded as one of main factors in the Polish efforts to regain independence. On November 6^th, 1916, under the headline: "Warsaw cheers promised freedom", The New York Times reported:

"The ceremony was short and simple. Precisely at noon, General Besseler, wearing the decorations granted for the reduction of Antwerp and the Polish fortresses mounted the dais of the gala ballroom of the old Jagiellonian Castle, and in the name of Germany's sovereign read the imperial manifesto in ringing, soldiery tones...
President Brudziński of the recently elected City Council, who is rector of the University of Warsaw, advanced before the dais and in the Polish tongue gave thanks to the imperial decree... President Brudziński, who was in plain civilian attire, without decorations, seemed to represent the spirit not of the ancient Poland and the Polish chivalry but of modern intellectual Poland."

The scientific careers of Brudziński and Moro followed a remarkably parallel path; aside from their bacteriological work under Escherich, both discovered neurological signs, now bearing their names. The Moro reflex in infants is a response to sudden loss of support and is believed to be the only unlearned fear in humans. Brudziński's career ended with his premature death, while the career of Moro, whose wife was of Jewish origin, was cut short by the Nazi rule.

References

Bertrand & Weisweiller (1906) Annales de l'Institut Pasteur Vol. 20, No. 12, p. 977 − 990.
Brudzinski J (1900) Ueber das Auftreten von Proteus vulgaris in Säuglingsstühlen nebst einemVersuch des Therapiemittels Darreichung von Bakterienculturen. Jahrbuch fur Kinderheilkunde und Physische Erziehung, Vol. 52, No. 3, p. 469 − 484. Berlin, October 10, 1900.
Carter EB (1919) Commercial cultures of Bulgarian Bacillus. Journal of Pharmaceutical Sciences 8, p. 179 − 183.
Cohendy & Michelson (1906) Compt Rend de la Soc Biol Vol. 17, 1906.
Grigoroff S (1905) Étude sur une lait fermentée comestible. Le "Kissélo mléko" de Bulgarie. Revue Médicale de la Suisse Romande. Genève. Georg & G., Libraires-Éditeurs. Librairie de l’Université.
Hanson P, Zinsser H (1914) A Text-book of Bacteriology: A Practical Treatise for Students and Practitioners of Medicine. D. Appleton, 1914, 769 pages.
Kendall AI (1911) Certain fundamental principles relating to the activity of bacteria in the intestinal tract, their relation to therapeutics. The Journal of Medical Research Vol. 25, Boston, Massachusetts, p. 117 − 187. Ed. Clarence Harold Ernst.
Massol M (1905) Rev Med de la Suisse Rom, p. 716.
Metchnikoff E (1908) Prolongation of Life. GP Putnam's Sons, New York 1908.
Moro E (1900) Ueber den Bacillus acidophilus n. spec.; Ein Beitrag zur Kenntnis der normalen Darmbacterien des Säuglings. Jahrbuch für Kinderheilkunde 52:38 − 55.
Pasteur L (1858) Ann de Chim et Phys s 3 1858 lii p. 404.
Poehl AW (1887) Bestimmung der Darmfäulniss durch Untersuchung des Harns. Jahresber., u. d. Fortschr. der Thier-Chem., 17, 277.
Rettger LF, Cheplin HA (1921) A Treatise on the Transformation of the Intestinal Flora: With Special Reference to the Implantation of Bacillus Acidophilus. Intestines. Yale University Press.
Shulman ST (2007) Theodor Escherich: the first pediatric infectious diseases physician? Clin Infect Dis. 45(8):1025 − 1029.
Stengel A (1908) Diseases of the intestines. in: Modern Medicine, Its Theory and Practice: In Original contributions by American and Foreign authors. Vol. 5, Eds. Sir William Osler, Thomas McCrae.
Tissier H (1900) Recherches sur la flore du intestinale normale et pathologique du nourrison. Thèse. Paris, 85 − 96.
Vikhanski L (2016) Immunity: How Elie Metchnikoff Changed the Course of Modern Medicine. Chicago Review Press.
New York Times, November 6, 1916 p.2: Warsaw cheers promised freedom.

Piotr Polaczek is at the Braun Labs, California Institute of Technology, Pasadena, California. Professor Józef Brudziński was the author's great-grandfather.