Small Things Considered

A Good Defense Is Worth Stealing

by Merry Youle

Add phage ICP1 to the list. Source.

One widely-used tactic for defense against phage and other mobile genetic elements is to deploy a CRISPR-Cas system (click here and here) to recognize and chop them into pieces. Based on sequenced genomes, 60% of Bacteria and 90% of Archaea have the wherewithal to dispatch invaders this way. But phages also have to protect themselves against enemies, including other mobile elements. Knowing a good thing when they see it—and they have seen it from the receiving end often—some phages have stolen the entire CRISPR-Cas structure and use it to inactivate genetic elements that would interfere with their replication.

What sorts of genetic elements challenge phage supremacy? Some bacteria harbor chromosomal islands that, like prophages, excise and replicate when induced by cellular stress or damage. When phage infection is what triggers the activation, the island is classified as a PICI, a phage-inducible chromosomal island. The ungrateful PICI sometimes then proceeds to interfere with phage replication. One subset of these PICIs includes the SaPIs, Staphylococcus aureus pathogenicity islands, that I introduced here and wrote more about here.

When a Good Peptide Deformylase Gets Better

by Merry Youle

In bacteria, the ribosome-associated peptide deformylase (PDF) removes the formyl group of the N-terminal formyl methionine of nascent proteins. This process is a prerequisite for the proteolytic removal of the unmasked methionine by methionine aminopeptidase (MAP). The enlargement shows PDF (cyan ribbon) bound to ribosomal protein L22 (magenta) next to the ribosomal tunnel exit (white star). The second interaction site of PDF on the ribosome, protein L32, is not visible in this view of the ribosome, which is sliced along the tunnel. The path of the nascent chain is indicated by yellow spheres. Source.

When a phage invades a host’s premises, it delivers only its genome and perhaps a few specialized proteins needed immediately upon arrival. Its plan is simply to supervise production. The host is relied on to provide not only the raw materials and energy, but also the production equipment needed to synthesize phage proteins and nucleic acids. This includes the machinery for further processing each protein as it leaves the ribosome. Sometimes, as in the case of the cyanophages, the host’s equipment isn’t quite enough.

Uniquely, proteins made on bacterial ribosomes, including those encoded by phage, start with an N-formylmethionine residue donated by a special initiator fMet-tRNA. As the N-terminus of the new peptide chain emerges from the ribosome exit tunnel, the formyl group is removed by the enzyme peptide deformylase (PDF); frequently the remaining N-methionine itself is also removed by a separate enzyme. Many proteins as they exit fold into their functional form with the help of a chaperone, the trigger factor (TF), stationed at the portal. Correct folding is essential for protein function, and the formyl group must be removed for proteins to fold correctly. Deletion of the PDF gene in E. coli is lethal. Likewise, actinonin, a naturally-occurring antibiotic that inhibits PDFs, also inhibits bacterial growth and disrupts photosynthesis in the chloroplasts of plants and green algae.

For most phages, a functionally adequate host PDF is something to be taken for granted. However, there is the danger that intense protein synthesis during phage infection can overwhelm the host’s supply of PDF, the upshot being improperly processed, non-functional proteins. This is more likely a problem for cyanophages. While all phages require the synthesis of various proteins used directly for their replication, cyanophages also depend on active synthesis of other proteins needed to maintain the host’s photosynthesis apparatus. This extra burden further taxes the host’s co-translational processing enzymes. Specifically, the high-turnover D1 protein of photosystem II must be replenished—the subject of an earlier post. For this problem, the cyanophages have a solution.

This story began when Sharon and colleagues searched the publicly-available marine metagenomes generated by the Global Ocean Survey (GOS), making use of their novel method to identify bacterial genes encoded in viral, not bacterial, genomes. Their yield was abundant; they found virally-encoded metabolic genes from 34 gene families. Of these 34, the most numerous were the PDFs, yielding 70 genes—all of which were from cyanophage genomes. Mapping of their data by Frank et al. located these phage PDFs throughout the oceans. Finding homologs of bacterial PDF genes in phage is one thing; demonstrating that these genes encode functional enzymes and are expressed during cyanophage infection is another. As a big step in that direction, Frank and colleagues demonstrated that cyanophage S-SSM7, a phage infecting the cyanobacterium Synechococcus WH8109, encodes a functional PDF with some phage-specific quirks.

Oddly Microbial: Prions

by Marcia Stone

Steven Schlozman’s book, soon to be a movie.

Electron micrograph of brain tissue from a monkey infected with the prion disease kuru. Between the rounded nuclei of the nerve cells (green) are tiny vacuole spaces typical of the disease’s dark spots. From the cover of the December 2012 issue of Microbe, the ASM Magazine. Image PHANIE/Photo Researchers, Inc.) Source.

Prions can turn their victims into zombies—they punch holes in their brains and steal their souls. The infected stumble about, might take a bite out of someone if hungry, certainly don’t think straight and, most important, lose their memories and with them the very essence of their humanity or “soul.” Zombie enthusiast and Harvard psychiatrist Steven Schlozman made the connection and has even written a science fiction novel, soon to be a film, about a prion-caused apocalypse called The Zombie Autopsies. In the book, influenza-like viruses with prion payloads, possibly concocted by mad scientists, cause a zombie pandemic that kill off a goodly portion of the planet’s humans; but not before the newly minted zombies stagger about trying to eat, and likely infecting, anyone they manage to fall into. Schlozman’s hypothetical disease, “ANSD” or Ataxic Neurodegenerative Satiety Deficiency Syndrome, is readily transmitted by coughing or sneezing.

OK. That neurologic prions turn people into “zombies” is a good analogy; sadly, that’s what seems to happen when Alzheimer’s and raft of other protein-instigated neurodegenerative diseases take hold. However, it’s unlikely that any amount of microbiological savvy could keep a tiny RNA virus schlepping around a prion or two afloat. And even if a few overloaded viruses manage to accidently plummet into an open nostril while struggling to stay aerosolized, it isn’t in the nature of prions to cause immediate disease. (More about prions from ASM.)

Biting the Hand That Clothes You

by Merry Youle

(Fig. 1.) This laughing gull is a kleptoparasite poised to steal food caught by the brown pelican. Since kleptoparasitism also includes the stealing of nest material from one animal by another, I would apply it also to the theft of capsid proteins from a helper phage by a SaPI. Source.

Troublesome strains of Staphylococcus aureus are often troublesome because they carry genes for superantigens and multiple antibiotic resistance. But don’t blame the bacteria. These genes are hitchhikers that arrived by horizontal gene transfer, embedded within mobile pathogenicity islands known as SaPIs. SaPIs are common; all S. aureus strains investigated so far carry at least one. They have also been found in other staph species and a few other Gram-positive genera. They have garnered much research attention because they rapidly move those clinically-significant genes from host to host. (They received some attention on this blog, too, several years ago.) The typical SaPI is composed of 15-17,000 base pairs of DNA that encode 18-22 proteins. Of those proteins, nine at most are required for the SaPI life cycle. This leaves plenty available to use to benefit or manipulate their host bacterium.

SaPIs resemble temperate phages in that they travel from host to host within a capsid and, upon infection, they choose between two options: they can replicate immediately or they can integrate into a specific site in the host chromosome and assume a quiescent state, held in check by a SaPI-encoded repressor. You might think of them as defective phages because they don’t encode the structural proteins required for their own capsids, relying instead on a “helper phage” to supply them. However, these kleptoparasites (see Fig. 1) are no more defective than every other sophisticated parasite that exploits another life form to provide some essential goods or services. It isn’t simple to be a successful parasite. Let’s consider how SaPIs do it.

Virus in the Room

I am the Lorax, I speak for the trees. I speak for the trees, for the trees have no tongues, and I'm asking you, sir, at the top of my lungs.
– from The Lorax by Dr. Seuss

by Welkin Johnson

Figure 1: A non-virus. Source.

As biologists, we divvy the biological realm up into domains using a formula that frankly, smacks of nepotism, bestowing three glorious domains upon our closest relatives—the Eucaryota, the Archaea, and the Bacteria—while committing an injustice to the so-called viruses, lumping them together in a miscellaneous catch-all category (“viruses” from Latin for poison and other noxious substances) with contemptible disregard for phylogeny or any true measure of diversity.

Imagine that viruses, like Dr. Seuss’s Truffula Trees, had a vocal advocate like The Lorax. Undoubtedly, through the agency of their outspoken mouthpiece, they would protest these gerrymandered borders and laugh at our skewed notions of biological diversity. After all (the viruses would argue), just consider the platypus, the coelacanth, the earthworm, and the bacillus. All these organisms have double-stranded DNA genomes, whose lengths all fall within roughly the same order of magnitude, which they replicate using evolutionarily customized versions of what amounts to the same basic enzymatic apparatus. How boring! How unimaginative! Now consider this (the viruses go on to say): the giant Mimivirus, 1256 nm of girth enfolding >1,000,000 base pairs of DNA, and the tiny Circovirus, with a mere 1,800 bases of single-stranded DNA tucked inside a 20nm-wide shell, are neither more nor less related to one another than either one is to an elephant! (For those who are not familiar with the elephant, it is a relative of the platypus, the coelacanth, the earthworm, and the bacillus)

Phee ɸ Phoh PhuZ: A Tale of Giant Phage with a Furtive Tubulin

by Daniel P. Haeusser

Fig. 1: Mattel’s 1985 “Battle Bones” toy assembled into a skeleton that organized and transported your Masters of the Universe action figures over vast rough terrains into combat. New research shows bacterial viruses (phage) encode cytoskeleton proteins that organize and transport phage DNA during phage replication in host cells.

A few years ago I attended an ASM Branch meeting where an investigator gave a talk about a metagenomic survey of oceanic bacteriophages. In typical fashion for this type of study, one slide listed dozens genes of note identified as being encoded in phage genomes. With surprise I noticed that one of these was ftsZ, which encodes the prokaryotic tubulin homolog responsible for cell division in most bacteria and several phyla of archaea. I wondered: Why on Earth would a phage contain a cytoskeletal protein? The ftsZ gene is even found in chloroplasts and the mitochondria of certain protists, but at least these organelles have the evolutionary history of having been independent membrane-bound cells. But with a virus there is no ‘cyto’ in which to place any ‘skeleton’. An obvious hypothesis is that the phage might make use of a tubulin-like protein within the host for it nefarious phage reproduction cycle – but in what way?

Enter two recent studies (Kraemer, et al. and Oliva, et al.) reporting phage-encoded tubulin homologs and the start of their characterization. A general model emerges for these proteins in directing phage DNA replication within host cells and maximizing phage proliferation. Intriguingly, the phages encoding these tubulin homologs have particularly large genomes, suggesting a possible correlation between giant genome size and the need for a phage-encoded cytoskeleton.

Revisiting BYOG: Bring Your Own Gene

by Merry Youle

This image, that accompanied the original BYOG
post, is a 17th century illustration of the Copernican
model of a heliocentric universe. Source.

For this, my last post as co-blogger, I chose to loop back to my very first post about my favorites among the many Small Things, the phages. That was BYOG: Bring Your Own Gene, published in March of 2008, recounting what was at the time a totally new story for me. Amazing! Those ingenious cyanophages brought along genes to keep the energy flowing for phage replication by maintaining active photosynthesis in their doomed cyanobacterial host.

The story back then was rather straightforward. The focus was on the genes that encode the two proteins at the core of photosystem II, D1, and D2, both proteins being subjected to photodamage. D1 in particular has an especially high turnover rate. The notion was that when the phage shuts down host protein synthesis, damaged D1 would not be replaced, photosynthesis would decline, and this would impact phage replication. Made sense.

Indeed, it was a good story. It had begun back in 2003 with the first report of photosynthesis genes encoded by a phage. That was followed in 2005 by a paper showing that these genes are actually expressed and translated into proteins during infection. By 2007 more such “host genes” had been identified in various phage genomes. Increasingly it was realized that these genes, though not essential for phage replication in the lab, provided fitness benefits to the phage. They were given the name auxiliary metabolic genes (with the acronym AMG). Since then, the array of AMGs has grown and includes genes involved in phosphate acquisition, nucleotide metabolism, and cytoskeletal construction (soon to be discussed on STC), as well as numerous genes involved in photosynthesis.

MicroRNAs and Retroviral Integrity

The tumor shown here was caused by bovine leukemia
virus (BLV). BLV’s mechanism of tumorigenesis has
long been a mystery, but researchers have recently
discovered a virus-encoded miRNA that may be the
culprit. Source.

by Jamie Henzy

Among retroviruses, the deltaretrovirus genus is something of a shady bunch, its members lurking in the shadows, causing trouble in the form of persistent infections that result in lymphomas and leukemias. At this time, we know of no endogenous sequences that might be their ancestors—the lone retroviral genus (of seven) for which this is the case, as if they’ve intentionally covered their tracks to elude the authorities. Fittingly, two members of this clan are emerging as particularly resourceful and crafty, exposing novel tricks in the viral arsenal.

These two are bovine leukemia virus (BLV), which infects cattle and causes B-cell tumors, and human T-lymphocytic virus (HTLV)-1, which causes T-cell tumors in its human hosts. Merry blogged about the stealth method of cell-to-cell spread employed by HTLV-1—how it uses immunological synapses to directly access target cells and avoid the exposure to immune defenses that a free virus particle would face. Not to be outdone by its sly associate, BLV has recently won the honor of being the first RNA virus convincingly shown to encode microRNAs.

Ehux: The Little Eukaryote with a Big History

by Jaime E. Zlamal

Coccolithovirus, a giant double-stranded
DNA virus, infects Ehux. The virus (pink)
was first observed in 1999 by W.H. Wilson
and was found to be a “giant-virus” having
472 protein-coding genes. Source.

There is an organism out there that is smaller than you, older than you, and that has a house that’s probably fancier than yours. Meet Emiliania huxleyi, or Ehux. Ehux is a miniscule coccolithophore, a beautiful single-celled alga that doesn’t get enough attention in the world of microbiology. The tendency of biologists to ignore this phytoplankton species belies its significance. In fact, Ehux has a penchant for being flashy—it has phenomenally beautiful plates covering it, known as coccoliths, intricate composites of calcium carbonate that when viewed under the microscope rival the most wondrous sculptures made by man. And they exist in huge quantities. The White Cliffs of Dover wouldn’t be so large, or for that matter, white, if it weren’t for this tiny architect. Ehux makes blooms near the surface of the oceans that are so big they can be seen from space! And it has its own web page.

A Table for Two

by Elio

TEMs of thin sections of infected V. vulnificus prey
cells. (A) Infected by phage only. (B) Infected by
BALO only. (C and D) Co-infection by both BALO
and phage. In (D) BALO did not change the shape
of the prey cell. B, C, and D show the bdelloplast,
the post-BALO infection structure with the predator
residing inside the prey cell. Bar = 500 nm. Source.

What fun, unless you are the one being preyed upon. Two predators, a phage and a “bacteriophagous” bacterium, simultaneously dine and reproduce in one host cell.

Doesn't this open a whole lot of questions, such as: How often does co-infection take place in the wild? Do the two predators “talk” to each other? Does this matter for the geochemical cycles? And, most important, who has first dibs?

Huan Chen and Henry Williams of Florida A&M University ask just such questions, using the marine bacterium Vibrio vulnificus as the prey, with both a phage and a BALO, Bdellovibrio-like organism (actually something called Cluster IX of Bacteriovorax) as predators. They mixed the participants in a predator/prey ratio of 1:1:1, let them work on each other, then looked at what was happening in thin sections under the electron microscope. The conclusion? The two predators do not seem to compete with one another, rather they both feast on the prey together. Both the invading bacterium and the phage multiply within the same host cell. This is a first.

Bdellovibrios and phages reproduce by very different mechanisms, but both require an intact host cell. Specifically, bdellovibrios penetrate the cell wall, reside in the periplasmic space, and shut off the host metabolic functions. They induce cell lysis after about 15 minutes, which is less than the time most phages require for their cycle. But the phage, it appears, is speedy enough to replicate in the time available. No doubt, there are some complex dynamics afoot.

This is a complicated business. Whereas each of the two predators has its own lifestyle with regards to cellular localization, reproductive strategy, and kinetics, co-infection makes special demands on each. Just how infection by BALO affects that of the phage and vice versa remains a fertile topic for investigation. Who says that things are simple in the oceans’ microbiome? The authors point out: Rather than the viruses and Bdellovibrio and like organisms competing for a single prey cell, both can survive in the same cell and successfully reproduce themselves. This is an especially valuable mechanism when the prey is in short supply, and the survival of the predators may be at stake.

The august (and relatively new) ASM journal mBio published this work in a section entitled Observations. This is a nice innovation for which the editors should be saluted.