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Reading for this lecture:

• Margulis L, Jorgensen JZ, Dolan S, Kolchinsky R, Rainey FA, & Lo S-C. 1998 The Arthromitis stage of Bacillus cereus: Intestinal symbionts of animals. Proc. Natl. Acad. Sci. USA 95:1236-1241.
• Jensen GB, Hansen BM, Eilenberg J and Mahillon J, 2003 Environ. Microbiol. The hidden lifestyles of Bacillus cereus and relatives.
• Huber H, Hohn MJ, Rachel R, Fuchs T Wimmer VC and Stetter KO. 2002 A new phylum of Archaea represented by a nanosized hyperthermophilic symbiont. Nature 417:63-67
• Waters E, et al. 2003 The genome of Nanoarchaeum equitaans: insights into early evolution and derived parasitism. Proc. Natl. Acad. Sci. USA 100:12984-12988

Symbiois, two or more organisms "living with" each other, usually refers to close and long-term interaction between organisms. Symbiosis can be ect- or endo-, and the symbiosis can be either facultive or obilgatory for each organism. Although symbiois is usually thought of in terms of mutually beneficial interactions, this is not necessarily the case; symbiosis can be beneficial to both (mutualistic), beneficial to one and not particularly harmful to the other (commensalistic) or beneficial to one and harmful to the other (parasitic). These relationships can even change with time; for example, many of the mutualistic organisms in your gut are also opportunistic parasites or pathogens. Where the line between these symbiotic relationship and more general ecological interdependecies lies is often unclear and subject to interpretation. For example, is a mosquito an ectoparasite? or a micropredator? What about a leech? or your brother-in-law?

There are all kinds of symbioses in Bacteria and Archaea. Symbioses between Bacteria and eukaryotes range from intimate and permanent obligatory symbioses (e.g. mitochondria), the wide range of surface and internal symbises, to pathogens on one hand and ecologically-critical nutriet cycling on the other. Symbioses between microbes can be highly specific and obigatory (Nanobacterium, below), syntrophic (organisms that exchange metabolic products, usually to mutual benefit), or as part of general nutrient cycling in an ecosystem.

Here we'll keep it simple with just a couple of xamples, one of the symbiosis of a bacterium ith an animal host, the other the parasitism of an archaeon by another archaeon.

Bacterial:animal symbiosis: Arthromitis

Some of the earliest good drawings of bacteria from microscopic observations describe the organism named Arthromitis in the gut contents of arthropods. Arthromitis are filamentous endospore-forming Bacteria, sometimes branched but usually attached at one end to either the gut epithelium or to various protists, fungi, &c. Although these were thoroughly studied and described in these early observations, there was no report of successful cultivation, and so none of the names were validated, and these organisms have generally been forgotten. So the question remained; what are these things called Arthromitis?

Disclamer:

Lynn Margulis (I'll call her Lynn, as if we were on a first-name basis) is a well-known scientist (and previously the wife of the late Carl Sagan), well-respected in some circles and dispised in others. She has been a very strong proponent of the role of symbiosis and cooperation as a driving force in evolution, a supporter of James Lovelock and the Gaia hypothesis. Earlier she strongly pushed the notion that chloroplasts and mitochondria were bacterial endosymbionts, well before this was proven by phylogenetic analysis of the organellar rDNA. Some believe she accepts (and encourages) inappropriate credit for the endosymbiont theory, which was put forth long before she was born and proven by molecular biologists. However, there is no doubt that she promoted this notion at a time when it was not generally accepted, and she was right. She has also strongly promoted the notion that eukaryotic flagella/cilia, and by extension the centriole, basal bodies, and microtubule organizing centers, and also the remnants of bacterial endosymbiosis. The visual evidence for this (Lynn is by nature a protist microscopist) is that there are protists in the guts of termites that have a covering of sprirochaetes attached by one end; these spirochaetes wave and move the protist around, and look an awful lot like cilia. However, cilia, centrioles, &c, lack DNA, and so the definitive rDNA test cannot be performed. Sadly, from her perspective, it turns out that the only homologs of tubulin that spirochaetes have is the same FtsZ that all Bacteria have, and it's no more similar to eukaryotic tubulin than any other FtsZ, nor are they organized into microtubules, much less anything like the complex arrangements of cilia.

Lynn is also a strong proponent of the related evolution-by-gene-transfer perspective; she is one of the folks I've refered to before who does not beleive that bacteria and archaea have species, but rather that existing organisms are a transient subsample of a single prokaryotic gene pool. Her dismissive attitude about microbial phylogenetics, and microbiologists in general, earns her no friends in some circles; particularly the scientific "offspring" of Carl Woese, which would include me. I heard her speak at an ISSOL (International Society for Study of the Origin of Life) meeting in Barcelona, and I have never before or since been angry at the end of a seminar. All the same, here's one of her papers, and it's an interesting one...

Arthropod intestines; the real habitat of Bacillus cereus?

Margulis L, Jorgensen JZ, Dolan S, Kolchinsky R, Rainey FA, & Lo S-C. 1998 The Arthromitis stage of Bacillus cereus: Intestinal symbionts of animals. Proc. Natl. Acad. Sci. USA 95:1236-1241.

Hypothesis: Arthromitis is the natural, gut symbiont form of the common soil organism Bacillus cereus.

The authors start with a simple string of observations:

1. if you look in bug guts, you see lots of filamentous spore-formers that have never been grown in culture
2. if you heat bug guts and plate it out, you get lots of Bacillus, but no Arthromitis
3. Bacillus is known to be a common bug symbiont

Until recently, most people have presumed that Arthromitis, as interesting as it might be, can't be cultivated and so isn't worth what would be hard work to study. But Lynn and her collegues (notice all the names in the acknowledgements; I think she's had undergraduate students working on this as a class project) know that organisms have complex life-cycles, and pleomorphism (different shapes) is more common in Bacteria than is usually appreciated. So they started with a simple hypothesis: if it's an aerobic (these guts are low oxygen but still aerobic) endospore-former, it must be related to Bacillus.

So they boiled bug gut contents for about 10 minutes (sound familiar?) and plated them out onto nutrient agar. The isolates they got look just like the Arthromitis seen in bug guts, but as these cultures are re-streaked and purified, they get changes in colony morphologies and the organisms begin to look more and more like Bacillus. In other words, the filaments go away and insetad you get individual cells and pairs. The resulting pure cultures are obligate aerobes, and are phenotypically indistinguishable from Bacillus cereus, and the ssu-rRNA sequnce confirms it; it's just B. cereus!

The culture conditions have a big role in the morphology of the cells; sporulation is facilitated by both light and oxygen. The inclusion of boiled bug gut extract in the media strongly favored growth as filaments.

Lynn proposes a life cycle based on microscopic observations in culture and from bug guts. Ingested spores germinate and swim around looking for an attachment site. They sprout attachment fibers (or a goo "thickening") to attach; this is important, otherwise they'll just pass right through the bug. The organism then grows as a filament, with spores being produced mostly from the distal end of the filament. These would then be excreted with the feces and sit in the soil waiting to be ingested by another host. Light and high O2 induce sporulation because this tells any unsporulated filaments that they're in a pile rather than in the bug. This life cycle isn't really so surprizing; this is essentially a symbiotic version of the life cycles of the insect pathogen Bacillus thuringensis and the mammal pathogen Bacillus anthracis, and these organisms are all so closely-related that they really should be considered the same species (along with B. mycoides). The amazing thing is that everything we thought we knew about Bacillus cereus turns out to be based on a complete misunderstanding of it's real habitat; even it's cellular morphology is to a large extent an artifact of domestication!

The one experiment that they didn't do, and as far as I know still haven't done, is an in situ hybridization of bug guts using a B. cereus-specific probe to confirm their results.

The Arthromitis species found in the guts of larger animals, such as mammals, turn out to be obligate anaerobes, as you might expect, and in turn are actually filamentous (under appropriate conditions) species of Clostridium.

Jensen GB, Hansen BM, Eilenberg J and Mahillon J, 2003 Environ. Microbiol. The hidden lifestyles of Bacillus cereus and relatives.

... is a great review of the life cycle of this subgroup of Bacillus - not the sporulation cycle, but the life style as related to symbiosis with their animal hosts.

The only known archaeal parasite

Huber H, Hohn MJ, Rachel R, Fuchs T Wimmer VC and Stetter KO. 2002 A new phylum of Archaea represented by a nanosized hyperthermophilic symbiont. Nature 417:63-67

Purpose : This is a monogram.

While isolating organisms from heated underwater gravel off the coast of Iceland, Karl Stetter's group (this is the same group that did Thermocrinus) isolated some new crenarchaea of the genus Igniococcus. They're interesting organisms in their own right, anerobic and autotrophic sulfur reducers and a hyperthermophiles, growing best at about 90C.

But while isolating these new Igniococcus species, they saw that in some of their cultures, the Igniococcus had tiny round blebs on them. In nicely growing cultures, these tiny blebs were nearly all stuck to the large Igniococcus cells, but in stationary phase cultures, there were lots of them floating free. These tiny things could be physically separated by either optical tweezers or standard "sterilizing" filters with a pore size of 0.45um. They could be stained with DAPI, which means they contained DNA, but could never be cultivated in the absence of Igniococcus (the Igniococcus could be grow separately from the small cocci, however). A pure (co-)culture was obtained by using optical twezers to pick out a single Igniococcus cell with a single one of these small blebs attached.

In EM pictures, the small blebs were about 400nm (0.4um) in diameter, and although they were smashed up against the Igniococcus cells, there were no specific attachment structures. They looked inside and out like a typical small archaeal cell. The small cells could be readily removed from their apparent host by mild sonication.

But when they used PCR and 16S rRNA analysis, the only sequence they got was that of Igniococcus, even using universal primers (the same ones we used in class). A fluorescent probe against this sequence lights up only the Igniococcus cells, not the blebs. However, using the less stringent method of Southern blotting, they could see that these blebs did contain 16S rRNA genes - they just wouldn't amplify with the usual primers. So they cloned and sequenced the 16S rRNA directly from the band on the Southern gel (i.e. the old-fashioned, pre-PCR way). The sequence is very unusual, thus the failure of the universal primers to amplify it. Of course they confirmed their data by making a FISH probe based on this sequence, that lights up only the blebs (which are, of course, small cocci), not the host. Trees of the sequence show it to be clearly archaeal, but apparently (there is some uncertainty here) not belonging to either the Crenarchaea nor the Euryarchaea; it is a 4th phylogenetic kind of archaeon. (The 3rd are the Korarchaea, which are known only from environmental 16S rRNA sequences.) They characterized the organism and named it Nanoarchaeum equitans.

N. equitans can only grow stuck to Igniococcus; no other host ill do, nor can it grow in media made from extracts of Igniococcus, nor in a co-culture with Igniococcus but physically separated from it by a dialysis membrane (these are old tricks). They say here that it doesn't seem to slow the growth of it's host, but in a later paper they correct this; it does slow the growth of it's host when present in larger numbers. So it's a parasite. This is the first hyperthermophilic symbiont known, and also the only known archaeal parasite. It is also the smallest cellular organism known (of course viruses are smaller, although it's in the same size range as poxvirus and a bit smaller than mimivirus).

So, how to learn more about it? Sequence the genome!

Waters E, et al. 2003 The genome of Nanoarchaeum equitaans: insights into early evolution and derived parasitism. Proc. Natl. Acad. Sci. USA 100:12984-12988

Purpose : To understand the nature of extremely minimalized genomes and the relationship between this parasite and its host.

The N. equitans genome consists of a single circular molecule of 490,885bp, and of course lacks a lot: no genes for lipid synthesis, no typical amino acid biosynthetic genes, no genes for glycolysis or gluconiogenesis, no TCA cycle genes, no cofactor/vitamin genes, no genes for nucleotide synthesis. It must get all of these from the host. Even the ATPase is a minimal version, presumably run in reverse to generate a proton gradient (at the expense of ATP) to run transport pumps needed to pump what it needs from the host. In may very well be an energy parasite, like Chlamydia. Basically, it just has the cytoskeleton and information processing genes; replication, transcription, translation, signal transduction, and the cell cycle.

An interesting aspect of the N. equitans genome is how disorganized it is. In most bacteria and archaea, genes are generally organized into operons with structurally and functionally related proteins encoded together. Not so in N. equitaans; not even the ribosomal proteins are together in operons (this is very highly conserved), and nor are the ribosomal RNAs (which are almost always encoded together in the order 16S-23S-5S).

Even the functional domains of enzymes are often encoded separately! The example they focus on in the genome paper is the alanlyl tRNA synthetase, which charges alanine onto it's tRNA. This protein has two domains, and in all other organisms, these two domains exist in the same polypeptide. But in N. equitans, the domains are encoded separately, on two genes distant in the genome, but if you mix the two encoded proteins, they come together and functional perfectly well.

The most interesting aspect of the genome to me is that they couldn't find the genes for 3 tRNAs; glu, his and trp. tRNA genes are usually easy to find, much easier than protein-encoding genes. Some Archaea have a unique form of intron in their tRNA (and a few other) genes, and 4 tRNAs have these introns, but the glu, his and trp tRNAs are AWOL. However, there are the usual number of codens in the N. equitans genome (for it's size) for these amino acids, so how do they do it?

The answer is that these tRNAs are encoded in pieces! In this genome paper, they mention that there are fragments of tRNA genes lying about, and in a later paper, it was shown that these pieces are transcribed, come together, and when they do, the flanking sequences create an intron-like sequence, which is spliced out in trans (i.e. from two peices, rather than out of a single peice as usual) by the normal splicing machinery to create functional tRNAs.

Questions for thought:

• How would you confirm that Arthromitis is really B. cereus?
  
  
• What is the difference in the cell biology of filamentous vs individual rod-shaped phases of an organism? What reasons can you suggest favoring filmentous growth in bug guts vs individual cells in culture?
  
  
• What other bacteria:animal symbioses can you think of? Archaeal:animal? What about plants? or protists?
  
  
• What do you suppose it might be in the bug guts that favors filamentous growth (remember that media with bug gut juice favors filamentous growth)? How would you determine what it really is?
  
  
• What bacterial:bacterial parasitisms do you know about?
  
  
• Can you give an example of syntrophism? What do each of the organisms get out of this?
  
  
• What advantage do you think organisms get out of arranging functionally-related genes into operons? How rigidly do different organisms do this? What kinds of genes are most commonly aranged into operons?
  
  
• If organizing genes into operons has an advantage, what about Nanoarchaeum? Why doesn't it use operons?
  
  
• Some people have suggested that the split genes (e.g. tRNAs, tRNA synthetases) in Nanoarchaeum represent a left-over of the primative state, before they were otherwise fused. What do you think about this? What is the evidence either way? How would you test this hypothesis?
  
  
• Can you think of any conceivable way Nanoarchaeum could dispense with any more genes?