Small Things Considered

Mycobacteria Make Spores?

by Peter Setlow

The title page of Robert Koch’s original article,
The Etiology of Tuberculosis, published in the
Berliner Klinische Wochenschrift, April 10, 1882.

Rarely does news come along that threatens to overturn our cherished beliefs. Since early after the discovery of tubercle bacilli by Robert Koch in 1882, the mycobacteria have been considered not to be spore formers. Now this is being challenged by a new report.

The genus Mycobacterium encompasses a number of major human pathogens including M. tuberculosis, among the most common pathogens carried by humans. A hallmark of M. tuberculosis infections is the ability of the organism to persist asymptomatically for long periods in the host, and then reappear and cause disease. The new paper by Ghosh et al. from Uppsala University in Sweden claims that members of some Mycobacterium species form spores that resemble those produced by well-studied Bacillus species. The authors worked primarily with M. marinum, a relatively fast-growing mycobacterium with a doubling time of 4-6 hours. In old cultures they observed forms that resembled spores not only in their morphology and their resistance, but also in their accumulation of dipicolinic acid, a signature molecule associated with spores of Bacillus species. Putative spores were also observed in old M. bovis cultures. In addition, they found that Mycobacterium genomes, including that of M. tuberculosis, contain homologs of genes important for sporulation of the model spore former, Bacillus subtilis. At least some of these genes were transcribed only in old M. marinum cultures.

Robert Koch (1843–1910),
discovered Mycobac-
terium tuberculosis, 1882.

These observations would certainly be a paradigm shift in Mycobacterium biology, particularly regarding our thoughts about the reasons for the latency of mycobacterial infections. Given the revolutionary implications of these conclusions, it is obviously imperative that a high standard of proof be applied to such work. This is of special concern for the mycobacteria, since these organisms grow more slowly than many Bacillus species (doubling times of ~30 min); consequently, the potential for contamination of old cultures with a much faster growing known spore former is a major concern. The authors have done a variety of control experiments to establish that the spores formed in their cultures are indeed from M. marinum rather than some contaminating known spore former, but skepticism about such a novel finding will certainly not be (and indeed should not be!) obviated by this first report. One would expect that this work will be rapidly replicated and extended in other labs, including analysis of the DNA in the purified spores and examination of additional Mycobacterium species for spore formation. If their conclusion that mycobacteria are indeed spore-formers holds up, it will truly be a paradigm shift. It will be fascinating to watch this story unfold, with either the confirmation or refutation of this initial report.

Peter Setlow is Professor of Molecular, Microbial and Structural Biology at the University of Connecticut in Storrs, CT. The sparkly young lady to his right is granddaughter Anne.

Life in a Big Mac

by Merry

IFs make it to the Mac. Image of P. caudatum
fed a mixture of prey bacteria (green) and IFs
of H. obtusa (yellow). The IFs (arrow) are in
the Mac, the prey bacteria in food vacuoles.
Bar: 25 μm. Source.

From the bacterial point of view, a paramecium is a microcosm offering several tempting niches for colonization. When you collect a paramecium from nature, it's likely to have hundreds—even thousands—of bacterial endosymbionts on board, in different locations. Some will likely be in the cytoplasm, others in other cellular compartments, including the nuclei. To remind you, the ciliates evolved their own particular approach to nuclear compartments. They have two kinds of nuclei, macronuclei (we'll call them Macs) and micronuclei (Mics), typically two or more of each. The Macs carry out all the RNA transcription during normal vegetative growth; the Mics transmit the germline genome to subsequent generations. Both the Mics and the Macs are hosts to specific symbionts. While locations in mitochondria and nuclei have been observed for bacterial pathogens of animal cells, they are relatively uncommon.

So far, nine endosymbionts are known to live within the nuclei of paramecia, about half in the Mics, half in the Macs. These nine species, α-proteobacteria all, have been lumped together into the genus Holospora because of their similar cytology and lifestyle. Their closest known relatives are Rickettsia and Ehrlichia. They all appear to be obligate endosymbionts, i.e., are not known to live outside their hosts. All are host-specific and nuclei-specific.

Life in a Mac has its perks, such as a goodly supply of nutrients and protection from cellular defenses, including lytic enzymes. But what's an endosymbiont to do when the host cell mates? During this process, the Macs are digested along with the symbionts. New Macs formed after conjugation are free of bacteria. This may be one reason why, unlike the cytoplasmic symbionts, all of these species are infectious, i.e., they are capable of horizontal transmission.

Structure of the IF of H. obtusa. Source.

Quite a bit is known about Holospora obtusa, a symbiont living in the Mac of P. caudatum. Its life cycle includes two stages. The reproductive form (RF) is a short rod, 1-3 μm by 0.5-1 μm, that grows and divides by binary fission within a Mac. Some RFs differentiate into an infectious form (IF), long rods 5 to 20 μm in length. This is a dramatic transformation, involving the replacement of 60% of their proteins, along with marked changes in cell structure.

Phase contrast micrograph of IFs outside the
host. Inset: After the IFs enter a Mac, they
undergo multiple divisions to give rise to
RFs. Three constrictions can be seen in the
IF on the left. Source.

Both forms coexist, intermingled, in the Mac until the host undergoes mitosis. At that time, some of the RFs move to the poles of the dividing Mac, thus ensuring that each daughter nucleus will inherit a share. Meanwhile, the IFs are destined to be released from the cells via a special, dedicated structure. They gather in a cluster in a central bridge that forms as the daughter Macs pull apart. This structure is seen only in symbiont-bearing paramecia, not in “cured” ones. As the daughter nuclei separate, the bridge breaks away from the Mac to form a membrane-bounded structure that travels through the cytoplasm to the cytoproct (the quasi-anus of ciliates), there to be discharged into the environment.

A recently ingested IF escaping from a phagosome
that is filled with other prey bacteria. Bar:
1.0 μm. Source.

The IFs can remain viable for weeks outside a host cell. They have a unique structure, the hallmark being a massive periplasm filled with proteins required for successful infection. IFs are ingested by paramecia along with other bacterial prey. But instead of being digested, they escape from the phagosome and journey—periplasm tip first—through the cytoplasm. They arrive at the Mac 30 minutes later. This process involves several fancy membrane fusions and evaginations. Host actin is also required. Once in the nucleus, the IF constricts transversely, dividing to form 4-10 RFs. The cycle is then repeated.

Many observations suggest a long shared history between H. obtusa and P. caudatum, the intimate communications between them during infection being one. Here are some others. The association seems to be quite specific. If you experimentally infect a different species of paramecium with H. obtusa, the IFs make it into the Mac, but within 24 hours they are deported to the cytoplasm, often in fragments, and "shat out" via the cytoproct. Although P. caudatum can get along just fine in the lab when cured of their endosymbionts, some natural isolates have H. obtusa on board, especially when collected in cold climates. In the lab, paramecia with H. obtusa endosymbionts do better in colder temperatures and also better handle rapid heating from 25 °C to 35 °C.

The symbionts alter host gene expression, up-regulating several genes and down-regulating others. Reproduction of the RFs in the Mac requires some proteins made by the host. Are these endosymbiont genes that have been transferred to the host genome? Initial estimates indicate a reduced genome size for the endosymbiont, about 1.7 Mb, but evidence of gene transfer must await genomic data.

Endosymbioses have a way of fascinating us. The very idea! Bacteria living within the organelles inside cells? When sketching a eukaryote cell, we draw a membrane around it, and around the nucleus and the other organellar compartments. These membranes mark serious boundaries. But endosymbionts remind us that such boundaries can be doorways—if you know the right incantation.

Fine Reading: In the Company of Ciliates

by Merry

Organisms such as ciliates that dine daily on bacteria run the risk of getting an infection. Indeed, ciliates—large, complex cells—are host in nature to gazillion different bacteria that they acquire primarily through phagocytosis at their "mouth." Resident bacteria are generally termed symbionts even though only a few of them have been studied enough to reveal the nature of their intimate relationship. The stories of those chosen few are recounted in an engrossing review by Hans-Dieter Görtz.

Some ciliated protozoa found in lakes and rivers. Those placed towards the top left are
typically found in the open water of lakes; those close to the centre at the base are all
anaerobic. The remainder are generally found in sediments and detritus, and attached to
submerged surfaces (e.g. aquatic animals and plants). All are drawn to scale (bar at the
right-hand side = 1 mm). Source.

These appear to generally be ancient relationships, on-going long enough that the partners have adapted to one another in complex ways. For the bacteria, the relationship tends to be obligatory. The ciliates, on the other hand, can mostly get along quite well when cured of their infections, at least under lab conditions. But, as usual, there are exceptions. One Paramecium has its needs for folic acid met by its Lyticum flagellatum symbiont; several freshwater Euplotes species die without their Polynucleobacter necessarius symbionts (aka "omikron particles"). And sometimes, as suggested in the case of the endonuclear symbionts to be featured in our next post, the symbionts may be beneficial under particular environmental conditions.

Some symbionts run offense for their host. Witness the Caedibacter, first known as "kappa particles," that convert their Paramecium hosts into "killers" that make toxins that kill "sensitives" of the same or other strains. Those that kill only during conjugation are termed "mate killers." Enough said. Others run defense, notably the Verrucomicrobium that resides as an ectosymbiont on Euplotidium and was featured in one of our first posts. Those associated with ciliates that live in hydrogen sulfide-rich anaerobic environments (such as in animal rumens or sewage sludge) are attracting more attention in this era of climate change because many of them produce methane.

Many more fascinating stories are undoubtedly yet to come. Optimistically, the author notes that the lag in their investigation may be partly due to the fact that intracellular bacteria could not be investigated with classical microbiological methods, since they cannot be grown outside their host cells. With the new powerful techniques for detection and phylogenetic classification such as polymerase chain reaction (PCR) and fluorescence in situ-hybridization (FISH) now available, this is changing. We can hardly wait.

Our thanks to Mark Martin for calling this fine paper to our attention.

The Viral Selenoprotein Theory

Indian paintbrush in bloom. These
plants absorb selenium from the soil
and concentrate it in their tissues.
The edible, sweet flowers were con-
sumed in moderation by various Amer-
indian tribes as a condiment. The high
selenium content, especially in roots
and green parts, makes it potentially
toxic in large quantities. Source.

by Chitra Rajakuberan*

Just when you thought that HIV has bared all its secrets, new ones come along. The HIV genome encodes 3 main polyproteins and six regulatory proteins. Like all other viruses, HIV relies extensively on the host for replication; of course, what sets it apart from most other viruses is its remarkable ability to integrate into the host genome and lie dormant for long periods of time.

Almost all the identified ORFs of HIV have been implicated in its pathogenesis. However, recent bioinformatics reports suggest that HIV may in fact encode for additional frame-shifted proteins. This is a fascinating story that didn't start with HIV. The frame-shift is brought about by RNA structures called pseudoknots. A pseudoknot is defined as an RNA secondary structure containing at least two stem-loop structures in which half of one stem is intercalated between the two halves of another stem. The pseudoknot structure was first recognized in the turnip yellow mosaic virus in 1982.

Example of a naturally occurring pseudo-
knot found in the RNA component of
human telomerase. Source.

Using software that predicts the existence of RNA pseudoknots, and hence the possibility of frame-shifted proteins, the existence of three new HIV proteins was deduced. These frame-shifts occur in the HIV protease, reverse transcriptase, and envelope protein sequences. Both the protease and reverse transcriptase are targets of existing anti-retroviral drugs, which make studying these new frame-shift proteins exceedingly important.

Most surprising is that all three frame-shift proteins are selenoproteins, i.e., they contain selenocysteine residues. Selenium is a trace element found in the soil and is a micronutrient essential for cellular activities. It is found in the active centers of reducing enzymes like glutathione peroxidase, thioreductase, and deiodinizing enzymes. Selenoproteins are found in almost all forms of life. In order to make selenoproteins, some UGA codons (otherwise termination codons) must be read as encoding selenocysteine, which requires an upstream RNA structure called the Selenocysteine Insertion Sequence (SECIS). Bioinformatics studies reveal the presence of these structures in HIV’s Long Terminal Repeats (LTRs).

Distribution of selenium in U.S. soils. Source.

A current hypothesis for the role played by selenium in HIV infection is that HIV selenoproteins sequester selenium and thus deprive the body of it, resulting in chronic selenium deficiency. Selenium deficiency can cause wasting and lowering of immunity and, most likely, the other classical symptoms of AIDS. Since selenium mainly functions as an antioxidant, the virus uses it to combat the cellular immunological responses like oxidative bursts. The viral selenoproteins may also be regulatory in nature and control HIV transcription. Preliminary studies indicate that HIV infection significantly lowers the selenoprotein level in T cells. Even though this hypothesis put forward by Will Taylor was initially for HIV, recent evidence seems to indicate that it could hold true for other retroviruses and also for other viruses, such as some strains of coxsackie viruses and even Ebola.

The viral-selenoprotein theory, though controversial, could, if proven, be a great turning point in virology. Not only would it highlight how efficient the viruses are in utilizing their genomes but it could also have a huge impact on the lives of the millions of HIV infected people all over the world. Just supplementing their diets with selenium might slow the progression to full-blown AIDS. Is this too simple to be true? The future will tell!

* Chitra was a student in the 2009 Integrative Microbiology graduate course at UCSD/SDSU. She is currently pursuing her Ph.D. studies in the lab of Roland Wolkowicz at San Diego State University.

Making Waves at Asilomar

by Elio

Asilomar breakers. Source.

It’s seldom that a bug has a negative effect on me. It's usually just the opposite: bugs make my day. But this time, some kind of bronchitis virus prevented me from going to a meeting that I would have truly loved to attend. The Integrated Microbial Biodiversity Program, under the umbrella of the Canadian Institute For Advanced Research (CIFAR), recently held their annual meeting in Asilomar, California. The location speaks to their willingness to transcend national barriers; the program, their transcending of conventional academic barriers, as well. The papers covering numerous ecological and evolutionary aspects of microbial life were presented by leading investigators from Canada and beyond. Included were such luminaries as Tom Cavalier-Smith, Ford Doolittle, Jonathan Eisen, Patrick Keeling, Forest Rohwer, and Curtis Suttle, to name just a few.

I had been asked to present stories from this blog, something that I have done with pleasure at several other venues. But this one would have been extra special. On all accounts, I missed out on what was truly my kind of meeting.

A few words about CIFAR from their website:

CIFAR has assembled a team of leading researchers from across Canada and around the world that is capable of taking on the monumental task of exploring this astounding universe of bacteria, viruses, and other microbes.

CIFAR’s Integrated Microbial Biodiversity program is transforming human understanding of the web of life, and changing the way we approach medicine and health, environmental sustainability, and evolutionary biology itself.

About Our Talmudic Questions

by Elio

Source.

We have now posted Talmudic Question #50, so this may be a time to reflect on this endeavor. True to our intent, these have been questions that have no definitive answers but most of the responses offered thoughtful conjectures. The pleasure resides in contemplating the question. What deeper meaning lies therein?

As an aside, the question “how many angels can dance on the head of a pin” may sound Talmudic but actually has a totally different origin, as discussed here.

The source of these Talmudic Questions? Notably, about one quarter were submitted by aficionados among our readers, and we are mighty glad for it. Those are among the very best. The rest arose from an inventory of questions carried along through the years, or that popped up at night during moments of wakefulness. As always, we have several more waiting in the wings. The well seems inexhaustible.

We hope that you enjoy this exercise and that you will share your answers with us. Better yet, consider sending us your own Talmudic questions. We know that there is at least one taking shape in each of you.

Talmudic Question #50

What do you think are the most interesting questions in present day microbiology?

Retrospective, June 2009

We present here a lightly annotated list that includes most of our posts from the past half year.

Source

Of Terms in Biology

We inaugurated this department in recent months. Terms in the spotlight so far:

• Eremacausis (here’s a new one for you)
• Metabolomics (its ins and outs)
• Obligate Parasite (which should have stirred a spirited controversy but barely did)
• Orthologs (to help you distinguish them from those other relations—paralogs)
• Panmictic (do you know this one?)

Question: Is this useful? Should we continue this?

Source

Microbial Diversity

The Microbe That Could Be Seen: Epulopiscium, that leviathan among bacteria, yields some of the secrets of its gigantism. With hundreds of thousands of copies of some of its genes, no wonder it can make so much of itself!

What You Didn’t Know About Janthinobacterium: Jenna Tabor-Godwin, Rhona Stuart, Rosa I. León Zayas, and Chitra Rajakuberan, students at San Diego State University and UC San Diego, tell us of a colorful bacterium that makes antibiotics that help its salamander host withstand fungal infection.

Source

Symbiosis

The Bacterium That Doesn't Know How To Tie Its Own Shoelaces: Carsonella is an endosymbiont of insects. With a genome of 160 kb, it barely qualifies as a bacterium—but it’s not yet an organelle. Strange-looking, too.

A Hot Happy Couple: The genome of Ignococcus hospitalis, the host to Nanoearchaeum equitans, has been sequenced, allowing a deep contemplation of the goings-on in this high temperature archaeal consortium.

Happy Together… Life of the Bacterial Consortium Chlorochromatium aggregatum: Mark Martin regales us with thoughts and facts about this misnamed lake-dwelling consortium composed of a chemoheterotrophic motile bacterium surrounded by a cadre of green-sulfur bacterial epibionts.

The Two Faces of Photorhabdus: This famed symbiont of nematodes is both a mutualist and a pathogen. It depends on which host you ask, the nematode or the insect. And it glows in the dark.

Source

The View From Here

Constructing a Synthetic Mycoplasma: Shmuel Razin recollected early thoughts on making a synthetic mycoplasma. Some things come around…

Fine Readings

Horizontal Gene Transfer in Eukaryotic Evolution: Patrick Keeling and Jeffrey Palmer discuss intriguing examples, exploring how they may have shaped the evolution of eukaryotes.

Emma Darwin: With all the bicentennial hoopla, did Mrs. Darwin get a fair shake? A broader view is presented by Mercé Piqueras.

Charles Darwin's sketch from 1837
captured how organisms evolve. Source.

Evolution

The Scandalous Bdelloid Rotifers: Rare among metazoans, they lack sex. But they have engaged in frequent horizontal gene transfer, which may have something to do with their extreme resistance to drying and irradiation.

Viruses and the Tree of Life: We reprinted Vincent Racaniello’s synopsis of a paper by Moreira and López-García that offered ten reasons why viruses should not be included in the Tree of Life.

The World is Pleiotropic: Our sales pitch for the notion that most biological macromolecules are multifunctional, using ribosomal proteins as examples.

Source

Pathogens

Coxiella Escapes from Cell! Coxiella burnetii, has been coaxed into growing in cell-free media. Scratch that one from the list of "obligate intracellular parasites."

A Pathogen's Swiss Army Knife: Maren von Köckritz-Blickwede discusses staph’s Protein A, a multifunctional protein that does a number of things to thwart the immune system.

Killer Prophage for Hire: Hydrogen peroxide made by pneumococci kills staphylococci by inducing their prophages.

30,000 Parasitoids Can't Be Wrong: These wasps deposit their eggs within other insect’s larvae. Why are the eggs not killed? This raises the issue of when a virus-like particle is not a virus.

Say, Brother, Can You Spare a DNA? Does DNA obtained by transformation help gonococci mend their ROS-damaged genome? And how do they take up useable genes but exclude potentially damaging foreign DNA?

Collateral Damage: Corals can be killed when folliculinid ciliates find them to be a convenient substrate on which to settle. No harm intended.

A Nuclear Family: In the dark world of hydrothermal vents, bathymodiolin mussels are host to essential bacterial symbionts, but also to a γ-proteobacterium free-loader. This critter invades the nuclei of gill cells where it reproduces, at the expense of the cell.

Source

Ecology

No Phosphorus? No Problem! (There’s More Than One Way to Skin a Phytoplankton): Cyanobacteria and small marine eukaryotes make do with little phosphorus in their environment by making sulpholipids instead.

A Fly in the Frozen Custard: 500,000 years is a long time to survive in glacial ice. But instead of being dormant, bacteria have been repairing their DNA all along. But were they actually frozen all that time?

The Secret Under the Ice: We reprinted a fine piece from the Spanish blog of Manuel Sanchez, Curiosidades de la Microbiología, on the Antarctic "blood falls," a curious outpouring of bright red ferric iron-rich material, a by-product of the work of sulfate-reducing bacteria.

Source

Odds & Ends

Music to the Tune of a Protein: Do you want to translate a protein sequence into music? Ask Stephen Zielinski.

Acoustic Mimicry: A rare non-microbial post. It’s about butterfly larvae that mimic the sounds made by queen ants.

Fine Reading: Emma Darwin

by Elio

Portrait of Emma Darwin
from the late 1830s by
George Richmond. Source.

Just when you thought that everything conceivable has been written about Charles Darwin on his bicentennial, a revealing perspective on his wife, Emma, appeared in the journal International Microbiology. Written by the distinguished science writer Mercé Piqueras, the article sheds light on many aspects of the relationship between Charles and Emma, including their correspondence while he was traveling on the Beagle. Of particular interest is the clash between her theistic beliefs and his agnosticism. They both learned to live with it.

Mercè also offers a rich eclectic blog in Catalan, La lectora corrent (The Common Reader, à la Virginia Woolf) where she shares her passion for language, science, and spreading the word. It’s also available in English and Spanish, but beware! It’s machine-translated.

The World Is Pleiotropic

by Elio

The bacterial ribosome is composed of three
different RNA molecules and more than 50
different proteins. Source.

So there! By this rash assertion I mean that in the biological world almost every macromolecular constituent is likely to function in more than just one way, i.e., is pleiotropic. One and the same protein may be enzymatic, regulatory, and structural; one nucleic acid, informational, structural, and regulatory; and so on. Clarence Jeffery called this “moonlighting.”

Most biologists know this, but how many think it’s a big deal? I do, based on little more than the belief that living things are parsimonious and make efficient use of their parts. Doesn’t it seem reasonable that our own 30,000 or so genes (a puny number, one would think) each encode many functions?

Linc Sonenshein put it well in a recent email: I believe that within 10 years, we will view all proteins as presumably multifunctional, that is, we will view monofunctional proteins as a rarity. It took us so long to come to this realization because we were raised on the one gene-one enzyme dogma and the great specificity of biological reactions, implying highly specialized functions that evolved to be the best they could be. It never occurred to us that cells (bacteria in particular) learned to do things that we hadn't thought of or that every protein is a compromise between the ideal for one of its functions and the less-than-ideal version that has the ability to carry out a second function.

Now for something more specific. But first, a disclaimer: “function” can be taken in many ways, cutting across biochemical and physiological categories. I don't want to get into this here, thus will use “multifunctionality” in a broad sense. I hope that’s OK with you.

A paragon of multifunctionality.
Source.

A recent review focuses on the multifunctionality of one particular set of proteins, those of the ribosomes. The numerous ribosomal proteins are juicy candidates. By definition, they are integral to the ribosomes and thus intimately associated with various RNAs and with translational activities. Evidence that they can also play a role in transcription has been accumulating for some time. Already by 2000, ribosomal proteins were front-and-center in a review entitled Proteins shared by the transcription and translation machinery.

The multiple functions of one ribosomal protein, S1, are an old story in microbiology. It’s been known since the mid 1970’s that the RNA phage Qβ uses a replicase composed of four subunits, one of which is indeed ribosomal protein S1. Two others are also borrowed from its E. coli host: the protein-synthesis elongation factors EF-Tu and EF-Ts. Thus only one of the four is phage-encoded. Protein S1 is a master of versatility. In E. coli it is required for ribosomes to recognize the translation initiation codon of most messenger RNAs; it stimulates a phage T4 endoribonuclease to inactivate some unneeded phage mRNAs by cleaving them in the middle of their Shine-Dalgarno sequence; and it seems to help out other ribonucleases, as well.

Other ribosomal proteins also moonlight. In E. coli their moonlighting balances the rate of their own synthesis with the rate of transcription of ribosomal RNAs. When ribosomal RNA is abundant, the proteins bind to it. But when the ribosomal RNA is in relatively short supply, they bind instead to their own mRNAs, thus acting as operon-specific translational repressors. For an example of a structural study of such interactions, click here. Some are involved in negative regulation. For example, ribosomal protein S2 serves as a negative regulator of the in vivo expression of both itself and a translation factor. The list goes on. Nearly 2/3 of the ribosomal proteins from the large (50S) ribosome subunit of E. coli have an RNA chaperone activity that ensures proper assembly of the ribosome. And four of the components of NUS, a transcription termination complex, are ribosomal proteins (S4, L3, L4 and L13). The list goes on and on…

Enough of bacterial fun. The examples of extra-ribosomal employment of ribosomal proteins extend to the eukaryotes, as well. The review we mentioned makes the case that ribosomal proteins also serve as sentinels for the self-evaluation of cellular health. Perturbation of ribosome synthesis frees ribosomal proteins to interface with the p53 system, leading to cell-cycle arrest or to apoptosis.

The list of instances is long, but it could be longer. The authors posit: Is this due to a lack of imaginative evolution by cells and viruses, or to a lack of imaginative experiments by molecular biologists?

Multifunctionality in proteins ought to give genomic annotators conniptions. Enough to drive them up a multifunctional wall.

A Nuclear Family

by Merry

(E) TEM of swollen nucleus with endonukes in longitudinal and cross-
section. The chromatin has been reduced to narrow remnants along
the nuclear membrane. (G) TEM of endonukes showing electron
dense particles distributed throughout the cells. Source.

Certain mussels called “bathymodiolins” are part of the spellbinding fauna of the dark world of oceanic hydrothermal vents and cold seeps. Similar to other metazoans in that realm, they rely on chemosynthetic bacteria for their nutrition. These mussels possess symbionts from two clades of γ-proteobacteria: chemoautotrophic sulfur oxidizers that fix CO₂ using sulfide or thiosulfate as their energy source, and methane oxidizers that use methane for both carbon and energy. The symbionts are well-housed in specialized cells (bacteriocytes) in the gills of the mussels where the constant flow of water brings the needed substrates to them. A pleasant mutualistic arrangement.

Recently researchers, using 16S rRNA analysis, found a member of a third clade of γ-proteobacteria in the gills, for which they proposed the descriptive name Candidatus Endonucleobacter bathymodioli. We'll refer to them as the endonukes, if it's okay with you. These are parasites with an atypical lifestyle. FISH probes specific for this bacterium found them most frequently in the gills, but only in symbiont-free intercalary cells (in between the bacteriocytes), and only in their nuclei. When an endonuke infects a nucleus, it feeds on the chromatin, and reproduces—ultimately killing the cell and releasing thousands of progeny (see figure). Since they do not infect the bacteriocytes, the host's nutrition is not curtailed and the mussels seem to be unaffected.

Developmental stages of the endonukes within gill tissues of the mussel, Bathymodiolus puteoserpentis. The images are projections of a stack of several two-dimensional layers, thus reflecting the overall three-dimensional structure in two dimensions. Green = endonuke. Blue = nucleus. Series shows the progression from a single short rod (G) to an unseptated ~20 μm filament (J), to stacks of shorter filaments (O). Continued reproduction yields an aggregate up to 30 μm in diameter, containing an estimated 10,000-80,000 bacteria. The nuclear membrane then ruptures, releasing the endonukes into water flowing through the gills. Source.

Rare are the bacterial parasites known to infect cell nuclei. Rickettsial α-proteobacteria occasionally invade nuclei of their arthropod or mammalian hosts, but typically are found in the cytoplasm. (In our earlier post about rickettsiae infecting mitochondria, we included a photo of rickettsiae in nuclei.) The authors suggest that the endonukes are far from rare. Already they identified them in mussels from 11 hydrothermal vents and cold seeps from around the globe. Members of their clade, known only by their 16S rDNA sequences, have been found to be associated with diverse marine animals including sponges, corals, clams, sea urchins, and fish. They might be associated with your next seafood combination plate.

Why the nucleus? Good food seems like one good reason; DNA is rich in nutrients. Should we coin a term? Which would you prefer: karyophagy? chromatophagia? or some other?

Say, Brother, Can You Spare a DNA?

by Merry

N. gonorrhoeae is a Gram-negative coccus,
usually seen in pairs with adjacent flattened
sides. Pili, extending several micrometers from
the cell surface, play a major role in adherence.
Source.

Mention horizontal gene transfer (HGT) in bacteria, and what comes to mind is the acquisition of new traits and capabilities across large evolutionary distances. Not so for the neisseriae. For them, HGT is a means to swap genes with other members of the species and to maintain the status quo in the face of assault on their genome by host defenses.

Infection by the human pathogen N. gonorrhoeae is met by the rapid recruitment of polymorphonuclear leukocytes (PMNs) to the site. PMNs are professional phagocytes that use a combination of antimicrobial proteins and reactive oxygen species (ROSs) to kill the engulfed invaders. Their ROS attack is effective against the gonococci and quickly kills the majority of them. However, a subpopulation not only survives within the phagosomes, it also replicates. A number of factors enable the gonococci to do this. An important one is having a means to repair the DNA damaged by the ROSs. This is where the HGT comes in.

A Gram stain of the urethral exudate from a
patient with gonorrhea. Source.

The neisseriae, including the gonococci, have a superb HGT capability that enables them to exchange DNA among themselves while excluding useless or potentially damaging foreign DNA. Genetic transformation (not transduction or conjugation) is their only known means of HGT, but it serves them well. As summarized by one researcher: Taken as a whole, the evidence suggests that an important benefit of transformation in N. gonorrhoeae is recombinational repair of oxidative DNA damages caused by oxidative attack by the host’s phagocytic cells.

False-colored scanning electron micrograph
of a human phagocyte and gonococci (green).
Source.

How does this work? Gonococci are both naturally competent and able to recognize their own DNA. Distributed throughout their genome are repeats of a conserved sequence called a DNA Uptake Signal (DUS). The minimum signal required for transformation is a 10-mer (5'-GCCGTCTGAA-3'). There are lots of these repeats, approximately 1900 of them per genome, which makes for roughly one every 1200 bp. Thus gonococci dedicate about 1% of their genome to these signals. Must be important.

DUSs are sprinkled throughout the chromosome. A third of them are located within genes; a quarter of all coding genes contain a DUS. The more DUSs present within a DNA fragment, the greater its transformation efficiency. DUSs occur more than twice as frequently in 3R genes—genes involved in DNA repair, replication, and recombination—which suggests that those genes would be picked up more often.

This much of the story is not unique to the neisseriae. There are more than 60 known naturally competent bacterial species. Selective DNA uptake has been observed among many of them. A search of eight sequenced genomes from the family Pasteurellaceae found conserved uptake sequences in all of them, and all eight show selective DNA uptake. Further investigation of Haemophilus influenzae found their uptake sequences also to be more frequent in their 3R genes.

DNA uptake mediated by a type IV pilus system in N.
gonorrhoeae. The pilins PilE and ComP are assembled to
form a competence pseudopilus. Double-stranded DNA is
channelled across the outer membrane through a pore formed
by the PilQ multimer, and periplasmic dsDNA is bound by
the membrane-anchored receptor ComE. Then, single-stranded
DNA enters the cytoplasm through the channel protein ComA,
while the other strand is degraded outside the cytoplasmic
membrane. Source.

The next step for the gonococci is to bring the recognized DNA into the cell using a type IV pilus system. Double-stranded DNA is channeled across the outer membrane. One strand then passes into the cytoplasm, the other is degraded in the periplasm. Once inside the cell, the DNA can be inserted into the recipient's chromosome by RecA-dependent recombination. RecA and DNA recombinational repair enzymes were demonstrated to provide one means of protection against oxidative damage caused by H₂O₂.

But this is only one side of the story—the uptake side. What about supply? It has long been known that some bacteria, including the pneumococci and streptococci, have ways of increasing the supply of closely-related DNA. They use quorum sensing so as to induce competence only when surrounded by many of their own kind, and combine this with fratricide to liberate potentially useful DNA into the medium. In contrast, actively growing neisseriae secrete chromosomal DNA into the medium without cell lysis, using a type IV secretion system (not to be confused with the unrelated type IV pilus). The secreted chromosomal DNA is readily taken up by recipient gonococci and incorporated into their genome by transformation.

Their type IV secretion mechanism is encoded by a genomic island that is related to the conjugative F-plasmid of E. coli and that has all the earmarks of having been acquired by HGT, albeit long ago. Eight separate mutations were made in genes located on the genomic island that were predicted to be required for type IV secretion. All were found to eliminate DNase I-sensitive DNA secretion and to greatly reduce transformation. As of 2005, this was the only DNA secretion system known that does not require direct cell-to-cell contact.

Apparently the gonococci make very frequent use of transformation, so frequent that, to quote one researcher, the entire genome is in flux, each gene undergoing recombination with imported DNA. This abundant transformation affects other genes, such as those involved in antigenic variation, while also providing a way to maintain core housekeeping genes damaged by ROS attack. Plus ça change, plus c'est la même chose.