An Arms Race at the Tiniest Scale

Defense and anti-defense against genetic attack by plasmids.

Bacteria have a pretty active sex life. And like for us, this involves defense and offense, in a complicated tango of genetic exchange. Only, for bacteria, conjugation mechanisms are overwhelmingly used for attack, even though they are also the conduit of great innovations like horizontal gene transfer from different species, and antibiotic resistance. The top priority of most bacteria most of the time is to defend against alien DNA, which most of the time are selfish genetic elements and viruses, and they have several mechanisms to do so.

Plasmids are a very common feature of bacteria, and fundamental to genetic engineering, as the primary form of manipulated DNA. Plasmids can be amplified to high copy number in bacteria, can be cut, altered, and ligated back together- the very essence of engineering. This dates me, but I remember preparing plasmids from bacterial cultures by cesium chloride isopycnic (high speed) centrifugation, after which the plasmid (marked by the poison ethidium) would end up swimming in the middle of the gradient, and have to be sucked out with a syringe, before further steps to clean up the purified DNA. It was a rather messy, expensive, uncertain, slow, and unsafe process. 

Summary of the current paper, outlining plasmid transfer, plasmid genome structure, and some components (genes and promoters) of the leading strand as it is transferred to target cells.

Anyhow, plasmids in the wild are typically aggressive genetic elements that carry (encode) some or all of the components needed to form an injection attack complex (i.e. type IV secretion system) that conjugates with other bacteria. Plasmids can also carry many other things, like antibiotic resistance, or stray genes from prior bacterial hosts, or transposing genetic elements. So most of the time, bacteria want to defend themselves against this kind of invasion, even though some of the time the gifts they bring can end up being highly beneficial.

One of their defenses is the restriction system. Another foundation of genetic engineering was and remains the restriction enzyme. These are enzymes that cut DNA at a particular sequence. One can imagine how useful it can be to have such specific scissors for these infinitesimal molecules, and most labs would have a freezer filled with a large library of such enzymes that could be used for breaking and reconstructing new (plasmid) molecules, and also for analyzing them by their pattern of "restriction" sites. In the wild, these enzymes are paired with DNA methylation enzymes that make the host genome invisible to the restriction enzyme, leaving only newly arrived alien DNA susceptible to cleavage, and thus destruction, by these enzymes. 

Another defense is the now-famous CRISPR system. Bacteria capture small bits of invading genomes, and, assuming they survive, knit them into special genetic modules in their own chromosomes. Then they express these small modules as RNAs that latch onto an enzyme called Cas9, (or related enzyme plus RNA systems), which are nucleases that are guided by these RNAs to cut and inactivate invading DNA that matches the RNA sequence. This system is noted as a sort of adaptive immune system that learns from experience, and passes its knowledge down genetically to future generations.

More detailed maps of three example plasmid genomes, showing the distribution of anti-defense and other genes at the leading edge of the genome (left end). Red and yellow colored genes are anti-defense genes of various kinds. The tiny arrows are promoters, each of the early-start kind that can operate on single-stranded DNA.

A recent paper discussed specialized anti-defenses that plasmids have against these and other bacterial defenses, including toxin/suicide systems and inducible stress responses. For it is naturally an arms race with innovation on both sides. Plasmids have an origin of replication where new DNA strands start, and this new strand is what is injected into the target cell. So there is a linear order of DNA and thus genes going into the target cell, head to tail. These researchers find that the anti-defense genes of plasmids tend to be bunched up at the head of the genome, where they get into the target cell first. These regions also have a special way to fold up their single-stranded DNA (into a cruciform shape) that forms immediate promoters that allow these "early" genes to be transcribed by the host RNA polymerase, before replication in the host cell has regenerated normal double-stranded DNA. 

The authors do a very wide survey of species and plasmids, and find that there is a wide variety of anti-defense genes, many of which have unknown functions. Among the known ones are: anti-restriction inhibitors, which directly bind and inactivate restriction enzymes, methyltransferases (MTase) that methylate restriction sites to make them look like host DNA, single strand binding proteins (SSB), which coat the single stranded DNA and protect it from detection as single stranded DNA, and may assist in repair after Cas9 or restriction cleavage, and antitoxins that inhibit the toxin systems or the SOS system. There are also anti-CRISPR proteins, which degrade the Cas9 enzyme, or inhibit its binding to target DNA.

Experiment to demonstrate the importance of being first... into the target cell. Targeting means that the bactierial cell has a CRISPR system targeting the incoming plasmid. acrIIA4 is an anti-CRISPR protein that effectively blocks cleavage of plasmid DNA by the CRISPR/Cas9 enzyme. The petri plate exhibits bacteria that can grow only if plasmid transfer was successful. See text for further details.

They finish with an elegant experiment that asks how important it is for the anti-defense gene to be at the front of the plasmid. The gene they chose was acrIIA4, which is an anti-CRISPR that very efficiently inhibits Cas9 cleavage of targeted DNA. The petri plate at top shows the growth of infected bacteria after infection by the experimental plasmid, selected for plasmid presence on antibiotic medium. The grey bar, in both diagrams is the control, which are cells whose CRISPR system does not target this plasmid. The plasmid transfers fine, and the cells grow fine. In contrast, if the cell's CRISPR system does target the plasmid, lack of acrIIA is fatal, (top), decreasing plasmid transfer by about three logs, or a thousand fold. Putting the acrIIA gene at the tail end of the plasmid (middle experiments) helps a little, and transfer is knocked down only a hundred fold. Putting the defense gene at the front of the plasmid, (leading), though, corrects plasmid defense almost fully, and transfer is down a few fold only. Lastly, if acrIIIA is placed in opposite orientation, (inverted), such that plasmid replication is needed before this gene can be expressed (it can't use the cruciform single-stranded promoter), it is virtually useless. So indeed, being first off the block when invading a target cell is critically important, since the host cell makes its defenses all the time- they are ready and waiting.

While most of these transfers are unwanted, sometimes plasmids integrate into bacterial genomes, and then when they start transferring themselves into other cells, they can bring along huge amounts of their host genomes. That starts to look like serious genetic exchange that begins to approximate sex in eukaryotes. So, some balance of defense, offense, and beneficial exchange is the lifeblood of ecology and evolution at this most ancient scale of life.

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A Very French Star Wars

Bruno Dumont's "The Empire" touches on the true meaning of Sci Fi. (Spoiler alerts!)

The online reviews are not very good, but to me, this film was both hilarious and profound. A bunch of scuzzy French villagers go about their normal business, fishing, arguing, flirting, driving around. Then, though the magic of acting, they betray another plot entirely. Some are extraterrestrials just taking human form, deeply engaged in some cosmic battle and sponsored by hulking space ships above, in the heavens. The kicker is that the space ship is topped by ... a gothic cathedral. At first, this just seems like a hilarious way to cut special effects expenses. Why not use the local cathedral to shoot the space ship interiors?!? But as you revolve all this in your mind, it starts to appear as though Dumont is making a more interesting point.

By the traditional theological story, aren't we all extra-terrestrials, trapped in human bodies, constantly fighting with the flesh and destined to return to a better realm? Conversely, isn't the standard science fiction story full of magical wonders and grand dramas and theologies? What if religion and science fiction are ultimately, as L. Ron Hubbard appreciated, the same thing? Transporter, resurrection; medical miracle, laying on of hands; Borg, Satan; tomato, tomahto. 

Unfortunately, sometimes the humanity takes charge, sex first and foremost fouling up the neat good vs bad dynamic. In the plot, neither side really does anything bad or good, reinforcing the absurdity of a film that comes off as a sort of French Terry Gilliam masterpiece. The "1"s come from the flying gothic cathedral and think of themselves as good, while the "0"s come from a flying Versailles, (which makes for a particularly ungainly space ship), and know they are demons. But they are all equally distracted by those human bodies.

The ending was, as far as I understood it, a disappointment. The armadas of mini-cathedral and mini-Versailles fighters are lined up for the final battle, à la Star Wars. But suddenly, they all get sucked into a wormhole, and ... end of film. It is almost as though Dumont holds out hope that there is a real deity, or at least higher being out there, to save humanity from this battle between these wonderfully absurdist extra-terrestrials. After the wind dies down, the local policemen and villagers are left to puzzle over the wreckage, and what these signs and wonders could have meant.

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Histones Require a Towtruck With a Winch

Motorized remodelers adjust and open up chromatin for gene expression.

Wouldn't it be nice if, on a stop-and-go congested highway, you could just plow through all the obstructing cars and go where you want? That is what polymerases get to do on our DNA, once they are set in motion. They plow right through chromatin, histones, DNA-binding transcription regulators, and everything else in their way. But getting them to that point is a different matter. Origins of replication need to be carefully cleared and set up. Promoters of genes need to be activated by the convergence of enhancer-binding proteins, promoter-binding proteins, and mediators of various kinds to get that RNA polymerase set on its path. And that is not so easy in a chromatin environment where the DNA is almost all covered by something, principally histones that wind up our DNA in tight little 146 base pair loops.

A basic schematic of nucleosome cores (yellow), composed of histone proteins, and how they wind up DNA and pack with each other.

So a class of "chromatin remodelers" have evolved that move histones around, and exchange histones in a way that facilitates transcription. It became apparent a couple of decades ago that regions of active transcription have altered histone composition, H2A.z/H3.3 instead of the regular H2A and H3 histones. These histones are looser, allowing regulatory proteins better access to the DNA as well as easing the passage of the polymerases. But how do they get there? It has also gradually become apparent that regulatory proteins come in different types, with some "pioneer" regulators able to bind in the midst of packed chromatin. These in turn recruit additional regulators, including enzymes that loosen up histones by chemically altering them with methyl, acetyl, and other modifications, and remodeling enzymes that push histones around, revealing DNA where other regulators can bind, and popping out conventional histones for more weakly-binding ones.

A few recent papers revealed the structure of a few of these remodeling enzymes, and compare them between yeast cells and human cells. There are a variety of these machines, which specialize things like nudging nucleosomes into regular spacing, or evicting/moving nucleosomes from particular regions, such as near transcription regulators, or exchanging nucleosomes in active regions. It is the latter that is being studied here. In yeast, this SWI/SNF family remodeler comes in two parts, NuA4, which is a histone acetylase, and SWR1, which uses ATP to winch out H2A and replace it with H2A.z. These protein complexes cooperate with each other and have related effects in opening local chromatin to be more transcription competent. In humans, these complexes are combined into one super-complex, TIP60-C, which weighs in at 1.8 million daltons, a dalton being the mass of one hydrogen atom. One can appreciate here, as in so many other details of biology, the nature of evolution- the tension between conservation and change.

Chromatin remodelers from yeast (top) and human (bottom). SWI/SNF is on the left, and RSC is on the right. Both of these remodelers have wide capabilities of moving or replacing nucleosomes. DNA is in orange, and the histone is at top, within the DNA coils. See text for further description.

At top are the structures of two yeast remodelers, SWI/SNF and RSC. At bottom are structures of the corresponding human remodelers, BAF and PBAF. One can appreciate how similar they are in overview, while being very different in detail. All of these remodelers function in detailed transcriptional control in collaboration with other regulators. The orange structure is the DNA wrapped around a nucleosome, while the large blobs at the bottom of these structures are relatively loose regions where they interact with other transcription regulators, which recruit them to the proper locations. The ATP-driven motor is shown at the top in green, and grabs tightly, with two RecA domains (that is to say, with two hands) to bits of the DNA circling the nucleosome. Given that the whole apparatus is anchored to the histone and other nearby structures, this enables the motor to pull on the DNA. It is pretty slow, moving only 1 or 2 base pairs per ATP used, but with enough copies (of these motors) and time, great things can be done. The structure in (b) indicates where the DNA enters (top) and where it gets pulled towards (bottom) as the motor works. This action can nudge the histone to some new location, relative to the DNA. Alternately, with other forms of anchoring, it can also pop the histone entirely off the DNA, and, since this large protein complex can bind alternative histone complexes, can bring in a new histone for exchange.  

Downloadable animation of nucleosome movement (source). 

Downloadable animation of overall assembly and nucleosome spacing activity (source).

So in short, what we have here is a DNA winch, which in various configurations can adjust, evict, or exchange nucleosomes, as directed by various signals. One signal is the sequence of the DNA itself, another is the standard spacing between chromosomes that is set by some of these motors that have a suitably sized extension, extending out to touch the next histone, and establishes the default nucleosome pattern, genome-wide. But more significant are the various pioneer regulators and histone modifiers that direct these motors to specific areas to reshape the local chromatin to control gene expression. Here is where the regulatory action is, and these proteins have been found to be determinants for cancer progression, and indeed are targets for some investigational anti-cancer drugs.

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An Uneasy Relationship With the Air

Review of Airborne, by Carl Zimmer. 

The pandemic was tough on everyone. But it had especially damaging effects on the political system, and on its relationship to the scientific community. Now the wingnuts are in charge, blowing up the health and research system, which obviously is not going to end well, whatever its defects and whatever their motivations.

While the scientific community had some astounding wins in this pandemic, in virus testing and vaccine production, there were also appalling misses. The US's first attempt at creating a test failed, at the most critical time. We were asleep at the wheel of public health, again at the earliest time, in controlling travel and quarantining travelers. But worst of all was the groupthink that resisted, tooth and nail, the aerosol nature of viral transmission of Covid. That is, at the core, what Zimmer's book is about, and it is a harrowing story.

He spends most of the book strolling through the long history of "aerobiology", which is to say, the study of microbes in the air. There are the fungal spores, the plant pests, the pollen, the vast amount of oceanic debris. But of most interest to us are the diseases, like tuberculosis, and anthrax. The field took a detour into biowarfare in the mid-20th century, from which it never really recovered, since so much of that science was secret, and in its shadow, the sporadic earlier public studies that looked carefully into disease transmission by aerosols were, sadly, forgotten. 

So it became a commonplace at the CDC and other public health entities, among all the so-called infectious disease specialists, that respiratory viruses like influenza, colds, and coronaviruses spread not by aerosols, but by contact, surfaces, and large droplets. This made infection control easy, (at least in principle), in that keeping a few feet away from sick people would be sufficient for safety, perhaps plus surgical masks in extreme situations. There was a curious disinterest in the older studies that had refuted this concept, and little interest in doing new ones, because "everyone knows" what the virus behavior is.

It is hard to explain all this in purely scientific terms. I think everyone knew at some level that the true nature of respiratory virus transmission was not well-understood, because we clearly had not managed to control it, either in residential or in hospital settings. It is hard to grapple with invisible things, and easy to settle into conventional, even mythical, trains of thought. First there were miasmas, then there were Koch's postulates and contact by fluids. It was hard to come full circle and realize that, yes, miasmas were sort of a thing after all, in the form of aerosols of infectious particles. It was also all too easy to say that little evidence supported aerosol spread, since the work that had been done had been forgotten, and the area was unfashionable for new work, given the conventional wisdom.

Even more significantly, the implications of aerosol spread of viruses are highly unpleasant, even frightening. The air we need every minute of our lives is suspect. It is a bit like the relationship we have with food- deeply conflicted and fraught, with fears, excesses, and rituals. One has to eat, but our food is full of psychological valences, possible poisons, cultural baggage, judgement, libraries full of advice. No one really wanted to go there for air as well. So I think scientists, even those calling themselves infectious disease specialists, (of all things), settled into a comfortable conventional wisdom, that droplets were the only game in town.

But what did this say about the larger research enterprise? What did it mean that, even while medical/bio research community was sequencing genomes and penetrating into obscure and complex regions of molecular biology, we had not done, or at least not appreciated and implemented, the most basic research of public health- how infectious diseases really spread, and how to protect people from them? It constituted gross negligence by the medical research community- no two ways about it. And that appears to have caused the public at large to question what on earth they were funding. A glorious enterprise of discovery, perhaps, but one that was not very focused on actual human health.

A timeline of research/policy

• Sprawling work on airborne infection in 1955. 

• Earliest glimmer in latest pandemic, May 2020. 

• Deeper followup, October 2020.

• Even deeper followup, August 2021. Later, 2023.

• Critics still can not believe aerosol spread, November, 2022.

• Hospital HVAC requirements currently go to MERV 8, not enough.

• Current CDC guidance mentions aerosols only from "procedures", not from people, though masks are recommended.

Aerosol spread of disease requires two things- that aerosols are produced, and that the infectious microbes remain infectious while in those aerosols. The former is clear enough. We sneeze, after all. Even normal breathing creates fine aerosols. The latter is where scientific doubt has been more common, since many viruses are not armored, but have loose coats and membranes derived from our own, delicate cells. Viruses like HIV don't survive in aerosols, and don't spread that way. But it turns out that Covid viruses have a half life of about two hours in aerosols. 

The implications of that are quite stunning. It means that viruses can hang around in the air for many hours. Indoor spaces with poor ventilation- which means practically all indoor spaces- can fill up with infectious particles from one or a few infected people, and be an invisible epidemic cloud. No wonder everyone eventually got Covid. 

What to do about it? Well, the earliest aerobiology experiments on infectious disease went directly to UV light disinfection, which is highly effective, and remains so today. But UV light is dangerous to us as well as microbes, so needs to be well-shielded. As part of an air handling system, though, UV light is an excellent solution. Additional research has found that far-UV, at 222 nm, is both effective against airborne microbes and safe for human eyes and skin, creating an outstanding way to clear the air. Another approach is HEPA filtration of air, either as part of an air handling / exchange system, or as stand-alone appliances. Another is better ventilation overall, bringing in more outside air, though that has high energy costs. Lastly, there are masks, which are only partially effective, and the place no one really wants to go. But given a lack of responsibility by those in charge of our built environment, masks are the lowest common denominator- the one thing we can all do to protect ourselves and others. And not just any mask, but the N95 high-quality filtration mask or respirator.

The pandemic threw some sharp light into our public institutions. We sequenced these viruses in a hurry, but couldn't figure out how they spread. We created vaccines in record time, but wasted untold effort and expense on cleaning surfaces, erecting plexiglass shields, and demanding masking, rather than taking responsibility for guarding and cleaning public air spaces in a more holistic way. It is a disconcerting record, and there remains quite a bit yet to do.

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Donald Trump is no Andrew Jackson

A few notes about the Jacksonian era.

One common historical touchpoint for our current epoch is the Jacksonian era, when a populist president presided over a significant increase in presidential power, carried into the White House by a ragtag rabble. Andrew Jackson stood against the elite power centers of the time, having been denied the presidency earlier by a shady deal that gave John Quincy Adams the office. Nor did he have much more love for the aristocrats of Virginia. He came from the backwoods of Tennessee, and a long career of fighting Indians as well as the English. Once in office, Jackson cleaned house and installed a patronage system that led to decades of increasing corruption, till the civil service was instituted. He also used the veto power, and made his cabinet secretaries subservient, to an unprecedented degree.

Jackson strengthened the party system and cultivated friendly media in a way that people at the time decried as divisive and dangerous. And, perhaps most strikingly, he oversaw the mass expulsion of Native Americans from the South. Jackson was a slaveowner and had no issue with the white supremacy of his day, whether against African Americans or Native Americans. Ironically, when France decided to not honor a treaty with the US, Jackson spared no effort to defend the nation's honor and rights. But when it came to the many treaties the US had signed with indigenous nations, many expressly meant for perpetuity, they were waved away like so much smoke.

On the other hand, Jackson was a successful general and businessman and won all the major battles of his presidency. And he was successful enough to anoint a successor, Martin van Buren. He was surprisingly eloquent and well-written and had a core set of principles that guided him and the nation. One principle was the importance of the constitution and the union. While previous presidents had thought the veto power should be confined to extreme legislative acts they regarded as unconstitutional, Jackson saw nothing in the constitution against using the veto on a policy basis, to weigh in on substantive issues as a popularly elected co-equal branch of government.

More importantly, he guided the nation through a nullification crisis with South Carolina with a sure hand. Always a hotbed of resistance and secession, South Carolina took particular issue with federal tariffs, which were set quite high to favor domestic industry. Industry generally located in the North. Jackson laid the groundwork for federal military intervention, promoted a tariff reduction, and issued a forceful and closely argued denunciation of "nullification" and secession that, in combination, squelched the movement of southern states against federal supremacy. This put off for a generation the crisis that Lincoln was fated to deal with.

One of Jackson's most interesting fights was against the Second Bank of the United States. Congress had chartered, from the Washington administration onwards, a national bank that was the sole interstate financial institution of the US. It was charged with facilitating the finances of the federal government, and with providing credit for internal improvements crossing state lines. But it was in essence a private bank that had only a fraction of its board appointed by the government and otherwise ran its business on a private basis as a commercial bank. In its opening years, it was generally undersized and not well run, and by the time of the second bank, had caused a couple of recessions due to its mismanagement. 

Finally, by the Jackson administration, it had come under competent management and was both expanding in all directions and doing a reasonable job of controlling the money supply and credit in the US, by limiting expansion of the state banks, (a significant source of opposition). It had, indeed, become the largest single financial institution in the world. But to Jackson, these were hardly points in its favor. He viewed it as a dangerous center of power, as though in our day JP Morgan were the only commercial bank allowed to do nation-wide business, with no competitors. The whole idea of a publicly-run central bank had not yet arisen at this time, and the national bank was more or less modeled on the Bank of England, which was a similar hybrid private entity. Unfortunately, instead of seeking reform of the national bank into a more modern and public-interest institution, Jackson pulled the only levers he had, which were to veto the rechartering of the Second National Bank, and then to follow that up with removing all federal deposits and putting them into state banks, effectively killing it. This had the unfortunate effect of dooming the US to almost a century of financial instability and poorly regulated banking. But on the whole, I am quite sympathetic to Jackson's position in killing the bank. It was a nascent form of anti-monopoly policy, which should have been taken up more systematically later in the century.

So, Jackson was very much of his time, not a visionary who could prepare the government for the vast growth in population, social institutions, and technology that were coming. But at the same time, he was not trying to drag the US backwards in time either. He did not cruelly run rampant through federal agencies, or foster international trade wars in search of a happier dream time of mediocre jobs and pay. The economic crisis that happened during his administration was not a tantrum he threw, but rather was caused by the national bank, as it consciously fostered a recession by withdrawing credit in an attempt to turn the people against Jackson. An attempt that failed because everyone knew what was going on, and which indeed showed the kind of power that Jackson was fighting against. Andrew Jackson did not view the federal government as an extortion racket or a throne from which bootlickers could be alternately fawned over and kicked in the teeth. He was thus, despite a few parallels, quite unlike the current occupant.

I am taking most of this material from an enjoyable biography by Jon Meacham. It is based mostly on correspondence, thus is quite chatty and focused on Jackson's domestic affairs. It is, conversely, frustratingly weak on the larger historical and policy issues of his day, particularly when it comes to the bank fight, which was so important for the country's future. 

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